Cellobiose Dehydrogenase

ABSTRACT

The present invention relates to cellobiose dehydrogenases (CDH) having glucose oxidation activity at a pH of 7.4 or above, modifications to modify the pH dependency of the enzymes activity, uses for these CDHs, in particular electrode sensors and electrochemical cells.

The present invention relates to cellobiose dehydrogenase (CDH) enzymes,modifications thereof and electrochemical uses.

Cellobiose dehydrogenase (EC 1.1.99.18, CDH) was first discovered 1974in the extracellular enzyme system of Phanerochaete chrysosporium andlater on in several other basidiomycete fungi. A special characteristicof this enzyme is its composition: the combination of a catalyticallyactive flavin domain, hosting a non-covalently bound FAD, and a haemdomain, with a haem b as a cofactor. Both domains are connected by aflexible linker. By its catalytic activity the natural substratecellobiose is oxidised in a reaction which reduces the FAD of the flavindomain. Subsequently, FAD can be reoxidised by the action of the haemdomain. The spectral characteristics of a typical CDH clearly show thepresence of both cofactors. Another characteristic described is thestrong glucose discrimination of all well characterised enzymes. Untilthe discovery of the ascomycete fungus Myriococcum thermophilum (Stoicaet al., 2005, Biosensors and Bioelectronics 20: 2010-2018; Harreither etal., 2007, Electroanalysis 19: 172-180), CDH was believed to stronglyinhibit the conversion of glucose (Henriksson et al., 1998, BiochimicaBiophysica Acta 1383: 48-54). Similarly, for a long time only CDHsexhibiting an acidic activity optimum were known, especially when thehaem domain is involved in catalysis as it depends on intramolecularelectron transfer (IET), which is necessary to transfer electrons viathe haem to the electron acceptor. This is the case with cytochrome c inenzymatic assays, as well as on electrode surfaces where the haem domainenables direct electron transfer (DET) to the electrode.

Electrochemical applications described in the literature are thedetection of cellobiose, cello-oligosaccharides, lactose and maltosesoluble cellodextrins, ortho- and para-diphenolic compounds (Lindgren etal., 1999, Analyst 124: 527-532) and catecholamines (Stoica et al.,2004, Analytical Chemistry, 76: 4690-4696) mostly by mediated electrontransfer (MET). So far the application in glucose biosensors based onthe direct electron transfer (DET) properties of CDH was prevented by i)a very low or no glucose turnover, ii) the acidic pH optimum of mostknown CDHs and iii) a bad performance of some CDHs on electrodes.

Although, one CDH with well functioning IET at neutral or alkaline pHvalues is known (from the fungus Humicola insolens), it was shown not toconvert glucose (Schou et al., 1998, Biochemical Journal 330: 565-571).One CDH currently known to convert glucose with significant turnovernumbers was found in cultures of Myriococcum thermophilum (Harreither etal., 2007, Electroanalysis 19: 172-180). However, this enzyme has anacidic pH optimum for the IET and shows no activity under physiologicalpH conditions (pH 7.4). Another obstruction is the sometimes badelectronic communication of a CDH with an electrode surface, like theHumicola insolens and Sclerotium rolfsii CDHs (Lindgren et al., 2001,Journal of Electroanalytical Chemistry 496: 76-81), which results invery low current densities and therefore low signals even with thenatural substrate cellobiose.

Harreither et al. (Electroanalysis, 19 (2-3) (2007): 172-180) discloseCDH direct electron transfer activity assays measured on an electrode.Activity at different pH values was determined with lactose orcellobiose as substrates. Although, glucose is accepted as a substrateat the pH optimum no information of the CDH activity on glucose at pH7.4 is given. As is shown in the comparative examples herein, theactivity of wild type CDH of M. thermophilum steeply decreases atneutral pH values above pH 5.5 and has no activity on glucose at pH 7.4.

Zamocky et al. (Prot. Expr. Pur. Acad. Press; 59 (2) (2007): 258-265)discloses the wild type M. thermophilum CDH and its DCIP activity whenusing citrate as substrate. The DCIP activity does not relate to the IEPactivity of the catalysis of carbohydrate oxidation reactions on anelectrode.

Database UniProt, Acc. No. A9XK88 discloses the wild type CDH sequenceof M. thermophilum.

U.S. Pat. No. 6,033,891 A discloses a CDH of Humicola insolens whichdoes not have a glucose oxidating activity.

Database EMBL, Acc. No. AF074951, AAY82220 and AAZ95701 providesequences of the CDH from Thielavia heterothallica. This enzyme does nothave an activity on glucose at pH 7.4.

Zamocky et al. (Current protein and peptide science, 7 (3) (2006);255-280) provide a review of CDHs of basidomycetes and ascomycetes.

Thus CDH activity on glucose under neutral conditions, which isnecessary for applications in e.g. physiological fluids is notsatisfying, in particular not for the electro-chemical measurement ofglucose or the generation of electricity in biofuel cells. It istherefore a goal of the present invention to provide an alternativeenzyme suitable to convert glucose at physiological pH ranges, inparticular on DET-based electrodes.

Therefore, in a first embodiment the present invention provides a CDHhaving glucose oxidation activity at a pH of 7.4 or above, selected froma CDH isolated from Chaetomium atrobrunneum, Corynascus thermophiles,Hypoxylon haematostroma, Neurospora crassa or Stachybotris bisbyi orbeing a modified CDH of Myriococcum thermophilum. According to theinvention it has been surprisingly found that certain CDHs have asuitable glucose oxidising activity under physiological pH conditions.Furthermore, the invention provides the modification of acidic CDHs toincrease their activity at pH 7.4 and above.

The term “cellobiose dehydrogenase” is defined herein as an enzymeconsisting of a flavin domain and a haem domain connected by a peptidelinker, which oxidises carbohydrates like its natural substratescellobiose and cello-oligosaccharides and others like lactose, maltoseand in particular glucose for preferred inventive uses. The reoxidationof the flavin domain cofactor can be achieved by direct oxidation bytwo-electron acceptors including quinones like 2,6-dichloroindophenol,o- or p-benzoquinone or derivatives thereof, methylene blue, methylenegreen, Meldola's blue or one-electron acceptors like potassiumferricyanide, ferricenium hexafluorophosphate, FeCl₃ or byintramolecular electron transfer (IET) to the haem domain cofactor andfurther to a terminal electron acceptor like cytochrome c (cyt c) or anelectrode surface.

The flavin domain of the CDH, which is responsible for the glucoseoxidation activity and the haem domain, responsible for the regenerationof the flavin domain, may have two different pH optima, such as in thecase of the natural CDH of Myriococcum thermophilum. In principle, thehaem domain can be bypassed by providing the flavin domain with oxidantssuch as 2,6-dichloroindophenol which can directly reoxidise the flavindomain without the haem domain. According to the present invention,however, it should be understand that the CDH has a glucose oxidationactivity at a pH of 7.4 by the action of both the flavin and the haemdomain (IET) as can e.g. be measured by the cyt c assay or bymeasurement after immobilisation on an electrode surface (DET—directelectron transfer—to the electrode). The haem domain acts asintermediate electron transmitter between cyt c and the flavin domain orbetween the electrode surface and the flavin domain, respectively.

The natural, wild-type CDH of M. thermophilum does not have theinventive glucose oxidation activity at a pH of 7.4 by action of boththe flavin and the haem domain. The present invention has now for thefirst time provided a modification of the CDH of M. thermophilum whichhas the desired glucose oxidation activity. This modification accordingto the present invention should now be understood in that the inventiveM. thermophilum CDH deviates from the wild-type M. thermophilum CDH bythe substantially increased glucose oxidation activity at a pH of 7.4.This modification can be facilitated by increasing the interactionbetween the flavin and the haem domains, e.g. by modifying specific keyamino acids responsible for that interaction as is further describedherein. Preferably increasing the interaction includes increasingcontacts or interaction energy between the domains. A prediction of suchmodifications can be easily made by computational methods using e.g.force field based energy calculations. Furthermore, the interaction canbe determined by measuring protein activity as described herein.Furthermore, it is possible to increase the pH dependency of the haemdomain to a more basic pH. The pH optimum of the flavin domain of thenatural CDH of M. thermophilum could in principle have the required pHproperties to oxidise glucose, as is e.g. shown in FIG. 2 f (bymeasurement of the 2,6-dichloroindophenol (DCIP) assay).

Also provided are enzyme preparations comprising novel CDHs. The term“enzyme” or “enzyme preparation” as used herein refers to a cellobiosedehydrogenase from a specified organism which is at least about 20%pure, preferably at least about 40% pure, even more preferably at leastabout 60% pure, even more preferably at least 80% pure and mostpreferably at least 90% pure as determined by polyacrylamide gelelectrophoresis (PAGE).

The present invention relates to cellobiose dehydrogenases isolated fromnovel producers or genetically engineered from existing proteinscaffolds, which are able to oxidise glucose more efficiently than thecurrently known cellobiose dehydrogenases. The kinetic constants of theenzymes responsible for this effect are a preferably lower K_(M) valueand a higher k_(cat) value for glucose than the currently characterisedenzymes (e.g., Phanerochaete chrysosporium CDH: K_(M)=1600 mM,k_(cat)=2.64 s⁻¹, Henriksson et al., 1998, Biochimica and BiophysicaActa 1383: 48-54; Humicola insolens CDH: no glucose conversion detected,Schou et al., 1998, Biochemical Journal 330: 565-571; Trametes villosaCDH: K_(M)=1300 mM, k_(cat)=1.92 s⁻¹, Ludwig et al., 2004, AppliedMicrobiology and Biotechnology 64: 213-222). In addition, the k_(m) andk_(cat) values for glucose oxidation of the inventive enzymes shall beat a pH of 7.4.

In a further aspect the present invention provides a cellobiosedehydrogenase of SEQ ID NO: 5 (Chaetomium atrobrunneum), SEQ ID NO: 7(Corynascus thermophilum), SEQ ID NO: 3 (Hypoxylon haematostroma), SEQID NO: 11 (Neurospora crassa) and SEQ ID NO: 9 (Stachybotrys bisbyi).Furthermore homologuous enzymes are provided having glucose oxidationactivity at a pH of 7.4 or above comprising an amino acid sequence beingat least 50%, preferably at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, in particular preferred at least 99%, identicalto any one of SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, SEQ ID NO: 9or SEQ ID NO: 11.

In preferred embodiments the CDH of Chaetomium atrobrunneum comprises anamino acid sequence of SEQ ID NO: 5. The CDH is readily available fromC. atrobrunneum using the isolation methods described herein. In afurther related aspect the present invention also provides a CDHcomprising an amino acid sequence of SEQ ID NO: 5, or an amino acidsequence being at least 83%, preferably at least 85%, at least 88%, atleast 90%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, in particular preferred at least99%, identical to SEQ ID NO: 5.

In preferred embodiments the CDH of Corynascus thermophilum comprises anamino acid sequence of SEQ ID NO: 7. The CDH is readily available fromC. thermophilum using the isolation methods described herein. In afurther related aspect the present invention also provides a CDHcomprising an amino acid sequence of SEQ ID NO: 7, or an amino acidsequence being at least 76%, preferably at least 78%, at least 80%, atleast 83%, at least 85%, at least 88%, at least 90%, at least 92%, atleast 94%, at least 95%, at least 98%, in particular preferred at least99%, identical to SEQ ID NO: 7.

In preferred embodiments the CDH of Hypoxylon haematostroma comprises anamino acid sequence of SEQ ID NO: 3. The CDH is readily available fromH. haematostroma using the isolation methods described herein. In afurther related aspect the present invention also provides a CDHcomprising an amino acid sequence of SEQ ID NO: 3, or an amino acidsequence being at least 68%, preferably at least 70%, at least 72%, atleast 74%, at least 76%, at least 78%, at least 80%, at least 80%, atleast 83%, at least 85%, at least 88%, at least 90%, at least 95%, atleast 98%, in particular preferred at least 99%, identical to SEQ ID NO:3.

In preferred embodiments the CDH of Neurospora crassa comprises an aminoacid sequence of SEQ ID NO: 11. The CDH is readily available from N.crassa using the isolation methods described herein. In a furtherrelated aspect the present invention also provides a CDH comprising anamino acid sequence of SEQ ID NO: 11, or an amino acid sequence being atleast 72%, preferably at least 74%, at least 76%, at least 78%, at least80%, at least 82%, at least 84%, at least 86%, at least 88%, at least90%, at least 92%, at least 95%, at least 98%, in particular preferredat least 99%, identical to SEQ ID NO: 11.

In preferred embodiments the CDH of Stachybotrys bisbyi comprises anamino acid sequence of SEQ ID NO: 9. The CDH is readily available fromS. bisbyi using the isolation methods described herein. In a furtherrelated aspect the present invention also provides a CDH comprising anamino acid sequence of SEQ ID NO: 9, or an amino acid sequence being atleast 59%, preferably at least 60%, at least 62%, at least 65%, at least70%, at least 72%, preferably at least 74%, at least 76%, at least 78%,at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, atleast 90%, at least 95%, at least 98%, in particular preferred at least99%, identical to SEQ ID NO: 9.

Preferably, a homologous or modified CDH of has 1, 2, 3, 4, 5, 6, 7, 8,9, 10, at least 11, at last 13, at least 15, at least 17, at least 20,at least 25, at least 30, at least 40, at least 50, at least 60, atleast 80, at least 100 and/or up to 100, up to 80, up to 60, up to 50,up to 40, up to 30, up to 30, up to 20, up to 15 amino acidsubstitutions, deletions, insertions or modifications, and any rangesbetween these values, as compared to any one of the CDHs of SEQ ID NOs3, 5, 7, 9 or 11.

The present invention provides novel sequences of CDHs from Chaetomiumatrobrunneum (SEQ ID NO: 5), Corynascus thermophilum (SEQ ID NO: 7),Hypoxylon haematostroma (SEQ ID NO: 3), Neurospora crassa (SEQ ID NO:11) and Stachybotrys bisbyi (SEQ ID NO: 9). The CDHs of these sequences,as well as homologues with at least 50% sequence identity thereto arenovel CDHs which also fulfill the inventive properties of having aglucose oxidation activity at a pH of 7.4. The modification ofhomologuous enzymes thereto with at least 50% sequence identity arepreferably of amino acids which do not lower the pH requirement on theglucose oxidation activity. Any such modification can easily be testedby a glucose oxidation test on e.g. an electrode surface or by a cyt cassay, using cyt c to reoxidise the haem and subsequently the flavindomain of the CDH. Homologues can be readily identified by sequencecomparisons such as by sequence alignment using publicly availabletools, such as BLASTP.

The inventive CDH may be a modified CDH of Myriococcum thermophilum,comprising a flavin and a haem domain, wherein electron transfer fromthe flavin to the haem domain is increased as compared to wild type CDHof M. thermophilum, preferably as measured by the cyt c assay. Thus theinvention relates to genetic engineering of a CDH to improve the enzymesactivity further in the direction of high IET under neutral or alkalinepH conditions. The methods for the modification may be any known in theart such as amino acid mutations, including amino acid substitutions,deletions or additions but also chemical modification/derivatisation ofamino acid side chains, in particular acidic amino acid side chains.

As mentioned above, the invention includes homologuous sequences to theinventive CDHs of SEQ ID NOs 1 (M. thermophilum—with furthermodifications to improve the pH dependency as mentioned above), SEQ IDNos. 3, 5, 7, 9 or 11 with at least 50%, preferably at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, preferably at least 90%, at least 95%, at least 98%, or atleast 99%, sequence identity to the above sequences of SEQ ID NOs 1, 3,5, 7, 9, or 11. Preferably the catalytic site has a minimum ofmodifications of e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino aciddeletions, substitutions or additions or is even exempted frommodifications as compared to the wild type catalytic sites. Thecatalytic site is from Phe 251 to Ala 287 (Rossman Fold, flavinbinding), Val 334 to Leu 345 and Met 724 to Asp 732 of the M.thermophilum sequence of SEQ ID NO: 1. Corresponding amino acids alsoexist for the CDHs of Chaetomium atrobrunneum (SEQ ID NO: 5), Corynascusthermophilum (SEQ ID NO: 7), Hypoxylon haematostroma (SEQ ID NO: 3),Neurospora crassa (SEQ ID NO: 11) and Stachybotrys bisbyi (SEQ ID NO:9).

Preferably any one of the inventive CDHs shows IET (the transfer fromelectrons from the flavin to the haem domain) under neutral, alkaline orpreferentially physiological (pH 7.4) pH conditions. To ensure asufficiently high electrocatalytic activity of the enzyme under thoseconditions the IET as measured with the cyt c assay at pH 7.4 should beat least about 10% of the value maximum IET value measured under acidicpH conditions, or more preferably about 20%, or more preferably about40%, or more preferably about 60%, or even more preferably about 80%, ormost preferably should the pH optimum of IET be already neutral oralkaline.

The cellobiose dehydrogenases show preferably a sufficiently high directelectron transfer (DET) rate from the enzyme to the electrode to obtaina sufficiently high response at low substrate concentrations for a lowdetection limit and a high sensitivity. Only enzymes exhibiting highenough a DET current at the applied overpotential of +300 mV vs. Ag|AgCl(in 0.1 M KCl) to result in a detection limit of glucose (the lowerlimit of the linear range of the electrode was defined as the detectionlimit) with a spectrographic graphite electrode setup below 4 mM (theusual blood glucose concentration in a healthy human is 4-7 mM).

Preferably, the inventive CDH has an glucose oxidation activity at a pHof 7.4 or above and comprises an amino acid sequence of amino acids 22to 828 of SEQ ID NO: 1 or an amino acid sequence being at least 50%,preferably at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 94%, atleast 95%, at least 96%, at least 98%, in particular preferred at least99%, identical to amino acids 22 to 828 of SEQ ID NO: 1, characterisedin that the amino acid sequence has at least one additional amino acidsubstitution, deletion or insertion to the sequence of SEQ ID NO:1increasing electron transfer from the flavin to the haem domain ascompared to wild type CDH of Myriococcum thermophilum of SEQ ID NO:1,preferably as measured by the cyt c assay. Given in SEQ ID NO: 1 is thewild type M. thermophilum CDH which does not have the required glucoseoxidation activity at a pH of 7.4. The sequence of SEQ ID NO: 1 hasfurther a signal peptide up to amino acid 21 which may not be present onthe final processed enzyme. It could now be shown that according to thepresent invention by a single (or more) amino acid substitution,deletion or insertion of the CDH with the final M. thermophilum CDHsequence the pH optimum of the glucose oxidation activity can be shiftedto a more basic pH, in particular to a physiologically relevant pH of7.4. Those skilled in the art can readily chose from possible amino acidmodifications given the extensive sequence and functional informationdepicted herein and furthermore, can without undue burden test themodified CDH by a simple cyt c assay as described herein. Preferably,the inventive modified CDH of M. thermophilum has 1, 2, 3, 4, 5, 6, 7,8, 9, 10, at least 11, at last 13, at least 15, at least 17, at least20, at least 25, at least 30, at least 40, at least 50, at least 60, atleast 80, at least 100 and/or up to 100, up to 80, up to 60, up to 50,up to 40, up to 30, up to 30, up to 20, up to 15 amino acidsubstitutions, deletions, insertions or modifications, and any rangesbetween these values.

In a further embodiment the electron transfer of the modified CDH isincreased by increasing electrostatic interaction between the flavin andthe haem domain, preferably at a pH of 7.4. The electrostaticinteraction can be increased by optimising charge interactions at pH 7.4of basic and acidic amino acids of the flavin and haem domain bysite-directed mutagenesis, electrostatic repulsion can be reduced orelectrostatic attraction increased. From the available sequence andstructural information those skilled in the art can readily chose from avast amount of such possible mutations which increase the interaction atpH 7.4, and preferably results in an increased activity in a cyt cassay.

As has been pointed out, the intramolecular electron transfer (IET) ratebetween the flavin domain and the haem domain depends heavily on the pH.E.g. in the basidiomycete Trametes villosa CDH IET is fast at pH 3.5,slows down significantly at pH 5.0, and is virtually absent above pH6.0. Contrary, the IET of the ascomycete H. insolens CDH is not affectedby alkaline conditions, having a pH optimum of around 8.0. From thekinetic data of Humicola insolens CDH an alkaline pH optimum for cyt creduction is obvious and the DET measured for that enzyme was highest atpH>7 and is thus an exception so far for CDHs. Interestingly, althoughan ascomycete CDH, Myriococcum thermophilum CDH has an IET behavioursimilar to basidiomycete enzymes. Preferably, the above amino acids aremodified in the M. thermophilum CDH to increase the activity at a pH of7.4 as measured in a cyt c assay. Furthermore it has been found thatthese amino acids are of particular interest for the activity of theenzyme. Amino acids corresponding to these amino acids in CDHs ofChaetomium atrobrunneum, Corynascus thermophiles, Hypoxylonhaematostroma, Neurospora crassa or Stachybotris bisbyi with preferablymodifications, according to the homologues to the sequences of the SEQIDs NO: 3, 5, 7, 9 and 11 in other amino acids than in thosecorresponding to the above amino acids of Myriococcum thermophilum ofSEQ ID NO: 1.

The changes in the above mentioned amino acids of SEQ ID NO: 1 in orderto increase the activity at pH 7.4 are preferably to increaseelectrostactic interaction between the flavin and the haem domain asmentioned above.

In particular preferred embodiments the modification of the CDH is amodification of the haem domain of any one of amino acids 90-100,115-124, 172-203, preferably of any one of amino acids 176, 179-182,195, 196, 198, 201 corresponding to the M. thermophilum CDH of SEQ IDNO: 1 and/or of the flavin domain of any one of amino acids 311-333,565-577, 623-625, 653-664, 696-723, preferably of any one of amino acids318, 325, 326, 328, 568, 571, 574, 575, 624, 654, 663, 702, 709, 712,717, correspond to the M. thermophilum CDH of SEQ ID NO: 1, or anycombination thereof.

Possible modifications include (i) the exchange of acidic amino acids byneutral (polar or apolar) residues (e.g. Ser, Thr, Ala) to decrease thenumber of negative charges and weaken the electrostatic force field ateither the haem or the flavin domain at neutral/alkaline pH values. (ii)The exchange of acidic amino acids by alkaline residues (Lys, Arg) toincrease the number of positive charges and weaken the electrostaticforce field at either the haem or the flavin domain at neutral/alkalinepH values, and (iii) the introduction of alkaline residues (Lys, Arg)instead of neutral residues (Hydrophobic or hydrophilic) to increase thenumber of positive charges and weaken the negative electrostatic forcefield at neutral/alkaline pH.

Particularly the modification may include an increase of positive chargein the of amino acids 172-203 corresponding to the M. thermophilum CDHof SEQ ID NO: 1, preferably of amino acid 181, in particular preferred aD181K mutation or D181R, and/or preferably of amino acid 198, inparticular preferred a D198K or D198N mutation, and/or a decrease of anegative charge of amino acids 565-577 corresponding to the M.thermophilum CDH of SEQ ID NO: 1, preferably of amino acid(s) 568 and/or571 and/or 574, in particular preferred a D568S and/or E571S mutationand/or D574S mutation, or any combination thereof, in particular thetriple mutation D568S/E571S/D574S.

In further embodiments the activity of the inventive CDH is a glucosedehydrogenase activity and may be an electrocatalytic oxidation ofglucose.

The inventive CDH may be isolated, in particular from Chaetomiumatrobrunneum, Corynascus thermophiles, Hypoxylon haematostroma,Neurospora crassa or Stachybotris bisbyi or any genetically modifiedcell to recombinantly express the inventive CDH. Isolation may beperformed by diafiltration and subsequent ion exchange chromatography bycollecting fractions with CDH activity. The CDH can be further purified,e.g. using hydrophobic interaction chromatography.

The inventive CDH may also comprise a linker or be a part of a fusionprotein. An inventive CDH polypeptide comprising the inventive sequencesmay be up to 500 kDa, up to 400 kDa, up to 300 kDa, up to 200 kDa oreven up to 150 kDa.

In another aspect the present invention provides a nucleic acid moleculeencoding a CDH of the invention. A preferred embodiment of the inventionis a nucleic acid molecule encoding a cellobiose dehydrogenase havingglucose oxidation activity at a pH of 7.4 or above and comprising a

-   -   nucleotide sequence of SEQ ID NOs 4, 6, 8, 10 or 12, or    -   the open reading frame of SEQ ID NOs 4, 6, 8, 10 or 12 or    -   a nucleotide sequence with at least 50%, preferably at least        55%, at least 60%, at least 65%, at least 70%, at least 75%, at        least 80%, at least 85%, at least 90%, at least 95%, at least        98%, in particular preferred at least 99%, identity to SEQ ID        NO: 2, 4, 6, 8, 10 or 12 or the open reading frame of SEQ ID        NOs: 2, 4, 6, 8, 10 or 12, further comprising a nucleotide        mutation, substitution, deletion or insertion, preferably a        codon mutation, substitution, deletion or insertion,    -   a nucleotide sequence that hybridizes with any one of SEQ ID NO:        2, 4, 6, 8, 10 or 12 under stringent condition.

“Stringent conditions” relate to hybridisation reactions under definedhybridisation conditions which is a function of factors as concentrationof salt or formamide in the hybridisation buffer, the temperature of thehybridisation and the posthybridisation wash conditions. Such conditionsare for example hybridisation at 68° C. in a standard SSC hybridisationbuffer containing 0.1% SDS followed by stringent washing in wash bufferat the same temperature. Stringent washing can be performed for exampleby two times washing with 2×SSC buffer followed by two wash steps with0.5×SSC buffer. Stringent hybridisation conditions will preferablyinvolve a temperature of 15° C. to 25° C. below the melting temperature(Tm), whereby the Tm of a hybridisation product of a nucleic acid probecan be calculated using a formula based on the g+c contained in thenucleic acids and that takes chain lengths into account, such as theformula Tm=81.5 to 16.6 (log [n⁺])+0.41 (% G+C)−600/N), wherein N=chainlength (Sambrook et al. (1989), which is incorporated herein byreference). In practice an estimated Tm for an oligonucleotide probe isoften confirmed and thus a person skilled in the art can calculate theTm for any chosen probe whose nucleotide sequence is known.

A nucleic acid sequence of M. thermophilum may be defined by the SEQ IDNO: 2 or the open reading frame of SEQ ID NO: 2, including homologs withat least 50%, preferably at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, in particular preferred at least 99%, identityto SEQ ID NO: 2 or the open reading frame of SEQ ID NO: 2, furthercomprising a codon mutation, substitution, deletion or insertion toencode a CDH with glucose oxidation activity at a pH of 7.4 or above,preferably of amino acids 22 to 828 of SEQ ID NO: 1 with an additionalamino acid substitution, deletion or insertion. Preferably the encodedCDH is of the amino acid sequence with the above mentioned amino acidmodification.

The inventive nucleic acid molecules encoding a CDH with glucoseoxidating activity at pH 7.4 may be isolated or purified. The inventivenucleic acid molecules, in particular their open reading frame may becomprised in a vector, preferably an expression or cloning vector. Aninventive nucleotide molecule might further contain regulatory elementssuch as promotors and enhancers. Such a nucleic acid molecule comprisingthe inventive sequences may consist of up to 1,000,000 nucleotides, upto 900,000 nucleotides, up to 800,000 nucleotides, up to 700,000nucleotides, up to 600,000 nucleotides, up to 500,000 nucleotides, up to400,000 nucleotides, up to 300,000 nucleotides, up to 200,000nucleotides, up to 100,000 nucleotides, up to 50,000 nucleotides or upto 25,000 nucleotides.

Particular benefits of the inventive CDHs are i) a high glucose turnoverrate, ii) sufficient activity of the flavin domain and IET at pH 7.4 andiii) good DET characteristics. It was further found that these CDHs andother known CDHs are in particular suitable as anodic material in abioelectrode as e.g. biosensor or biofuel cell.

One approach was to identify CDHs of different fungal strains from thephylum of ascomycota. It was found that some CDHs, e.g. from Chaetomiumatrobrunneum, Corynascus thermophiles, Hypoxylon haematostroma,Neurospora crassa or Stachybotris bisbyi are able to convert glucosewith high turnover rates at pH 7.4 and have additionally very good DETproperties. Some of the found CDHs are new per se as mentioned above. Afurther aspect of the present invention is the use of all CDHs describedherein on an electrode, in an electrochemical cell, in particular in abiosensor to measure glucose, preferably at a physiological pH such aspH 7.4.

Furthermore it was found that CDH from M. thermophilum, which is knownto oxidise glucose and have good DET properties but shows no IET at pHvalues above pH 7.0, can be genetically engineered to increase the pHrange of the IET to more alkaline conditions by means of site-directedmutagenesis. These CDHs were particularly suitable for electrochemicaldevices.

Thus, in a further aspect the present invention provides an electrodecomprising an immobilised CDH having glucose oxidation activity at a pHof 7.4 or above and having at least 10%, at least 12%, at least 14%, atleast 16%, preferably at least 18%, at least 19%, in particularpreferred at least 20%, at least 21%, or even at least 22%, at least23%, at least 24%, at least 25%, at least 26%, at least 27%, at least28%, at least 29% or at least 30% glucose, lactose or cellobioseoxidising activity at a pH of 7.4 as compared to their maximal activityat a lower pH as determined by the cyt c assay. Preferably theimmobilised CDH also has a glucose oxidation activity at a pH of 7.4 orabove and having at least 10%, at least 12%, preferably at least 18%, atleast 19%, in particular preferred at least 20%, at least 21%, or evenat least 22%, at least 23%, at least 24%, at least 25%, at least 26%, atleast 27%, at least 28%, at least 29% or at least 30%, glucose, lactoseor cellobiose oxidising activity at a pH of 7.4 as compared to theirmaximal activity at a lower pH as determined by a 2,6-dichloroindophenol(DCIP) assay. An electrode is generally a conducting surface, e.g.suitable for an electro-chemical element.

The activity measurement by either the cyt c or DCIP assay can bereadily facilitated in a model system. The CDH can be directly used withthe substrate (glucose but also lactose or cellobiose) and a reoxidisingagent being either cyt c for reoxidation at the haem domain or DCIP forreoxidation at the flavin domain. The CDH is tested at a pH of 7.4 inany suitable buffer, e.g. a potassium or natrium phosphate buffer. Todetermine the maximum of the CDH activity, the activity is continuouslymeasured at different pH values, e.g. ranging from pH 3 to pH 7.4 orhigher and determining the maximum activity. The pH of 7.4 is thencompared with this maximum activity and should have the requiredactivity fraction mentioned above. The activity can e.g. be given asabsolute values in U/mg or as relative values. Preferably, the inventiveCDH has the required activity portion as compared to the maximumactivity in both a cyt c and a DCIP assay. These activity valuespreferably also apply to the new CDHs described above, as such,independent of their fixation on an electrode. Preferably, the assay todetermine the inventive CDH on the electrode is performed by glucoseoxidation. Alternatively, also using lactose is possible.

The electrode may be of any material suitable to immobilise the CDH,e.g. carbon such as graphite, glassy carbon, boron doped diamond, goldelectrodes modified with promoters e.g., thiols, screen-printedelectrodes, screen printed electrodes containing carbon nanotubes(single or multi-walled). It may contain other nanoparticles to increasethe specific surface area. Particular uses of the inventive electrodesare in the provision of biosensors and enzymatic biofuel cells, morespecifically to glucose biosensors and glucose oxidizing biofuel cellanodes using the direct electron transfer properties (DET) of cellobiosedehydrogenase (CDH) to measure the glucose concentration at neutral,alkaline or, preferentially, physiological pH (in human body fluids,e.g., 7.4 in blood) or use glucose for the generation of an electriccurrent in biofuel cells under the same pH conditions.

In particular preferred embodiments the specific activity for glucoseoxidation by using the cyt c assay at pH 7.4 is higher than 0.5 U/mg,preferably at least 0.6 U/mg, at least 0.7 U/mg, at least 0.8 U/mg, atleast 0.9 U/mg, at least 1 U/mg, or at least 1.2 U/mg CDH, or a currentdensity higher than 80 nA/cm² at pH 7.4.

In further preferred embodiments the apparent K_(M) value of the CDH forglucose in solution (DCIP assays at optimum activity) is lower than 1.7M, preferably lower than 1.5, lower than 1.2, preferably lower than 1 Mor, when measured on electrodes an apparent K_(M) value below 200 mM,preferably below 150 mM.

In another embodiment the present invention provides an electrode,wherein the CDH is of Chaetomium atrobrunneum, Corynascus thermophilus,Hypoxylon haematostroma, Neurospora crassa or Stachybotris bisbyi or amodified CDH of Myriococcum thermophilum with an increased activity atpH of 7.4 as defined in above, or homologues with certain sequenceidentities, amino acid modifications, etc. as defined above.

On the electrode, the CDH may be immobilised by adsorption, preferablyalso physical entrapment, complex formation, preferably via anadditional complexing linker, covalent binding, in particular crosslinking, or ionic linkage and/or the immobilized cellobiosedehydrogenase can be cross-linked, in particular by bifunctional agents,to increase stability or activity. It has been shown that crosslinkingwith bifunctional agents, such as agents with two reactive groups makinga connection with the CDH, can stabilize the CDH and even increase itsactivity on graphite electrodes measurable by amperometric methodsdescribed herein. This advantage can lead to an increased sensitivityand lowering the detection limit for glucose. Such a cross-linking agentis e.g. glutaraldehyde or any other dialdehydes.

The electrodes might be used in form of a single electrode or electrodestacks. More specifically, the application of these enzymes is in(bio)electrochemical devices such as glucose biosensors or biofuel cellsanodes. The electrode may be used as biosensor or as biofuel cell anode.

In another aspect the present invention provides an electro-chemicalcell comprising an electrode as described above as an anodic element anda cathodic element.

In preferred embodiments of the electrochemical cell the anodic fluidcan be glucose containing solution. Preferably the electrode is suitablefor measurement in blood, serum and other body fluids.

The electrochemical cell may further comprise a solution of at least pH6.0, preferably at least pH 6.5 or at least pH 6.7, in particularpreferred at least pH 7.0, even more preferred at least pH 7.1, or atleast pH 7.2, or at least pH 7.3, especially preferred at least 7.4, asanodic fluid.

According to another aspect a method of detecting or quanti-fyingglucose in a sample is provided comprising

-   -   providing a CDH having glucose oxidation activity at a pH of 7.4        or above,    -   contacting a fluid sample having a pH of at least 6.0,        preferably at least 6.5, or at least 6.7, more preferred at        least 7.0, at least 7.1, at least 7.2, in particular preferred        at least 7.3, especially preferred at least 7.4, with the CDH,        and    -   detecting an oxidation of glucose of the sample by the CDH.

Preferably the oxidation is detected electrochemically, preferably withan immobilised CDH on an electrode, in particular preferred as definedabove.

One of the world-wide leading causes of death and disability isdiabetes. The diagnosis and management of diabetes mellitus requirescontinuous monitoring of blood glucose levels. Amperometric enzymeelectrodes, based on glucose oxidase, play an increasingly importantrole and have been a target of substantial research. Most sensors areused for individual, daily diabetes monitoring, but the demand forcontinuous in vivo monitoring of patients is also significant. Real-timemeasurements are highly desired in intensive care units, during surgery,or for the management of diabetes, where rapid biochemical changes canbe missed by discrete measurements. Such monitoring requiresminiaturized, biocompatible, and stable sensors. Although research hasreached the level of short-term implantation, an implantable glucosesensor possessing long-term stability has not yet been realised. Besidesthe obvious biocompatability challenge, some sensors are prone to errorsdue to low oxygen tension or electroactive interferences. Thirdgeneration biosensors depend on enzymes that are able to permit directelectron transfer (DET) between the electrode material and the redoxactive centre. Usually this is hindered by the encapsulation of theredox center by the protein structure. However, as has been shownherein, the inventive CDH can exhibit electrical communication withelectrode supports.

In certain embodiments the CDH has at least 10%, or at least 12%,preferably at least 14%, or at least 16%, in particular preferred atleast 18%, or at least 20%, at least 21%, or even at least 22%, at least23%, at least 24%, at least 25%, at least 26%, at least 27%, at least28%, at least 29%, or at least 30%, glucose, lactose or cellobioseoxidising activity at a pH of 7.4 as compared to the maximal activity ata pH below 7.4 as determined by a cyt c assay and/or DCIP assay.

The fluid sample may be any fluid which potentially comprises glucose,including blood, serum and other body fluids.

In particularly preferred embodiments the CDH is of Chaetomiumatrobrunneum, Corynascus thermophiles, Hypoxylon haematostroma,Neurospora crassa or Stachybotris bisbyi or a modified CDH ofMyriococcum thermophilum with an increased activity at pH of 7.4 asdefined above.

The cellobiose dehydrogenase of the present invention may be obtainedfrom microorganisms of any genus. For purposes of the present inventionthe term “obtained from” as used herein in connection with a givensource shall mean that the enzyme is produced by the source or by a cellin which the nucleic acid sequence of the cellobiose dehydrogenase genefrom the source has been inserted. The enzyme or its nucleic acidsequence may be obtained from any fungal source and in a preferredembodiment from the genus Chaetomium, Corynascus, Hypoxylon,Myriococcum, Neurospora or Stachybotrys. In a more preferred embodimentthe enzymes or the nucleic acid sequences are obtained from the speciesChaetomium atrobrunneum, Corynascus thermophilus, Hypoxylonhaematostroma, Myriococcum thermophilum, Neurospora crassa orStachybotrys bisbyi.

In the most preferred embodiment the enzymes or the nucleic acidsequences are obtained from the strains Chaetomium atrobrunneum CBS238.71, Corynascus thermophilus CBS 405.69, Hypoxylon haematostroma CBS255.63, Myriococcum thermophilum CBS 208.89, Neurospora crassa DSMZ 2968or Stachybotrys bisbyi DSMZ 63042.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states and other taxonomicequivalents, e.g., anamorphs, regardless the species name by which theyare known. Those skilled in the art will readily recognise the identityof appropriate equivalents.

It is understood that one of skills in the art may engineer thementioned or other cellobiose dehydrogenases to obtain the outlinedspecifications of the enzymes and enzyme variants described herein toobtain modified enzymes using the principles outlined herein like therational approach via site-directed mutagenesis or directed evolutionapproaches (e.g., gene shuffling, error-prone PCR) and subsequentscreening of the generated diversity. The techniques to introduce amutation into the nucleic acid sequence to exchange one nucleotide foranother nucleotide with the aim to exchange one amino acid for anotherin the resulting protein may be accomplished by site-directedmutagenesis using any of the methods known in the art.

The present invention is further illustrated by the following figuresand examples without being restricted thereto.

FIGURES

FIG. 1 gives the codon-optimised nucleotide sequence (SEQ ID NO: 2) andthe corresponding amino acid sequence (SEQ ID NO: 1) of Myriococcumthermophilum CDH used for site-directed mutagenesis. Non-limitingpreferred mutations sites are indicated by “*” and particularnon-limiting preferred sites are marked by “+”.

FIG. 2 shows the pH profiles of screened ascomycete CDHs Chaetomiumatrobrunneum CDH, 2.a; Corynascus thermophiles CDH, 2.b; Hypoxylonhaematostroma CDH, 2.c; Neurospora crassa CDH, 2.d; Stachybotrys bisbyiCDH, 2.e; Myriococcum thermophilum CDH, 2.f; and the geneticallyengineered enzyme variants Myriococcum thermophilum CDH variant D181K,2.g; Myriococcum thermophilum CDH variant D547S/E550S, 2.h using lactoseand a soluble electron acceptor, 2,6-dichloroindophenol, (DCIP, dotted,grey lines) or cyt c (solid, black lines) as substrates and 50 mMcitrate-phosphate buffer (pH 3.0-8.0). FIG. 2 i shows the relativedirect electron transfer current of wild type Myriococcum thermophilumCDH with 5 mM glucose.

FIG. 3 is a sequence alignment of amino acid sequences of CDHs fromChaetomium atrobrunneum (SEQ ID NO: 5), Corynascus thermophilum (SEQ IDNO: 7), Hypoxylon haematostroma (SEQ ID NO: 3), Myriococcum thermophilum(SEQ ID NO: 1), Neurospora crassa (SEQ ID NO: 11) and Stachybotrysbisbyi (SEQ ID NO: 9).

FIG. 4 shows a setup of the wall jet electrode and auxiliaryinstruments. The sensor assembly (A) was continuously flushed withbuffer and samples were applied through an ultrafast injection valve.The obtained current at a potential of 300 mV was recorded. The flow-jetsystem (A) consisted of a carbon working electrode (WE), a platincounter electrode (CE) and a silver reference electrode (RE) connectedto a potentiostat.

FIG. 5: Measurement setup of the flow-cell system

EXAMPLES Example 1 Materials

Chemicals used in buffers and fermentation media were commercialproducts and at least of analytical grade if not otherwise stated.Peptone from meat and microcrystalline cellulose were from VWRInternational (Vienna, Austria), alpha-cellulose from Sigma-Aldrich(Vienna, Austria). Substrates for kinetic studies were lactose, glucose,2,6-dichloroindophenol (DCIP) and cytochrome c from horse heart(cyt c)from Sigma-Aldrich in the highest grade of purity available. Bufferswere prepared using water purified and deionised (18 Me) with a Milli-Qsystem (Millipore, Bedford, Mass., USA), fermentation media containedreversed osmosis water (0.1 Me).

Example 2 Enzyme Assays

Enzymatic activity of cellobiose dehydrogenase was detected by twoassays. The DCIP assay, measuring the activity of the flavin domain wasperformed by measuring the time-dependent reduction of 300 μM DCIP in 50mM citrate-phosphate buffer at the indicated pH (3.0-8.0), containing 30mM lactose at 520 nm and 30° C. The absorption coefficient for DCIP ispH dependent but differs at 520 nm only about 3% within pH 3.0 to 8.0and was determined to be 6.8 mM⁻¹ cm⁻¹ (Karapetyan et al., 2005 Journalof Biotechnology 121: 34-48).

Alternatively, enzymatic activity was determined by the reduction ofcytochrome c at 30° C. and 550 nm (cyt c, c₅₅₀=19.6 mM⁻¹ cm⁻¹,Canevascini et al., 1991, European Journal of Biochemistry 198: 43-52)in an assay containing 20 μM cyt c and 30 mM lactose, which specificallydetects the activity of the whole enzyme (flavin and haem domain). Thecyt c assay gives thereby also a measure of the efficiency of theintramolecular electron transfer (IET) between both domains as anindication of the enzyme's response on electrodes in a pH range of 3.0to 8.0 (50 mM sodium citrate-phosphate buffer). For the detection ofactivity with glucose the above mentioned assays were used, but lactosewas exchanged for 100 mM glucose.

One unit of enzymatic activity was defined as the amount of enzyme thatoxidises 1 μmol of lactose per min under the assay conditions. Lactosewas chosen instead of the natural substrate cellobiose, as it shows nosubstrate inhibition with CDH. The reaction stoichiometry withcarbohydrates is 1 for the two-electron acceptor DCIP, but 2 for theone-electron acceptor cyt c.

Example 3 Enzyme Kinetics

Carbohydrate stock solutions used for measuring activity and kineticconstants with the DCIP and cyt c assays were prepared in theappropriate buffer several hours before the experiment to allowmutarotation to reach equilibrium. pH profiles were determined using 50mM citrate-phosphate buffer (3.0-8.0). To ensure an assay temperature of30° C. the cuvettes were incubated in a thermostated chamber for atleast 20 min. After the measurement, the pH was again checked in thecuvettes. Kinetic constants were calculated by fitting the observed datato the Henri-Michaelis-Menten equation or to the adapted model forsubstrate inhibition using nonlinear least-squares regression and theprogram SigmaPlot (Systat Software, San Jose, Calif., USA).

Example 4 Protein Characterisation

The protein concentration was determined by the dye-staining method ofBradford using a pre-fabricated assay from Bio-Rad LaboratoriesHercules, Calif., USA) and bovine serum albumin as standard according tothe manufacturers recommendations.

For spectral characterisation apparently homogeneous CDH (in theoxidised state) was diluted to an absorption of −1 at 280 nm and thespectrum from 260 to 700 nm taken with an Hitachi U3000spectrophotometer (Tokyo, Japan). After reduction with lactose (finalconcentration 1 mM) the reduced spectrum was taken.

For electrophoretic characterisation SDS-PAGE was carried out on aHoefer SE 260 Mighty Small II vertical electrophoresis unit. Gels(10.5×10 cm; 10% T, 2.7% C) were cast and run according to themanufacturers' modifications of the Laemmli system. Isoelectric focusingin the range of pH 2.5 to 6.5 was performed on a Multiphor II systemusing precast, dry gels rehydrated with Ampholytes (GE HealthcareBiosiences, Vienna, Austria). Protein bands on the SDS-PAGE were stainedwith silver, bands on the IEF gel with Coomassie blue R-250, accordingto the instructions.

Example 5 Screening for Suitable Cellobiose Dehydrogenases

Fungal strains (Chaetomium atrobrunneum CBS 238.71, Corynascusthermophilus CBS 405.69, Hypoxylon haematostroma CBS 255.63, Myriococcumthermophilum CBS 208.89, Neurospora crassa DSMZ 2968 and Stachybotrysbisbyi DSMZ 63042) were obtained from the Centraalbureau voorSchimmelcultures (CBS, Utrecht, The Netherlands) and Deutsche Sammlungvon Mikroorganismen and Zellkulturen (DSMZ, Braunschweig, Germany) infreeze dried or actively growing form on agar slants and wereperiodically subcultured on potato dextrose agar (PDA) plates. Freshlyinoculated agar plates were grown at 25 or 30° C., depending on thepublished growth temperatures of the cultures until reaching a diameterof 5 cm and then used to inoculate shaking flasks. The medium used forsubmersed cultures contained (per litre): 20 g of alpha-cellulose, 5 gof peptone from meat and 0.3 ml of a trace element solution. The traceelement solution contained (per litre): 1 g of ZnSC₄.7H₂O, 0.3 g ofMnCl₂.4H₂O, 3 g of H₃BO₃, 2 g of CoCl₂.6H₂O, 0.1 g of CuSC₄.5H₂O, 0.2 gof NiCl₂.6H₂O, 4 ml of H₂SO₄ (Sachslehner et al., 1997, AppliedBiochemistry and Biotechnology 6365: 189-201). For the cultivation inshaking flasks, 1 L Erlenmeyer flasks were filled with 0.3 L of medium.After sterilisation the flasks were inoculated with 3 cm² of finely cutmycelium from PDA plates and incubated in a rotary shaker (110 rpm,eccentricity=1.25 cm) at 25 or 30° C. Samples were taken regularly andthe production of CDH was monitored.

Example 6 CDH Production and Purification from Fungal Sources

CDH production was performed in up to 16 parallel shaking flask culturesper strain using identical conditions as in the screening procedure.Cultures were harvested on the day exhibiting maximum cyt c activity.The culture supernatant was separated from residual cellulose and fungalbiomass by centrifugation (20 min, 6000×g) and concentrated anddiafiltrated using a polyethersulfone hollow fibre cross-flow modulewith a 10 kDa cut-off (Microza UF module SLP-1053, Pall Corporation)until a conductivity of 2 mS cm⁻¹ was reached. The concentrated enzymepreparation was applied to a DEAE Sepharose column (chromatographyequipment from GE Healthcare Biosciences) mounted on an AKTA Explorersystem and equilibrated with 50 mM sodium acetate buffer, pH 5.5. Thecolumn was eluted with a linear salt gradient (0 to 0.5 M NaCl in thesame buffer) in 10 column volumes (CV). Fractions with a high specificCDH activity were pooled, saturated ammonium sulphate solution wasslowly added at 4° C. to 20% final saturation and applied to aPHE-Source column equilibrated with 100 mM sodium acetate buffer, pH 5.5containing (NH₄)₂SO₄ (20% saturation) and 0.2 M NaCl. The column waseluted with a linear gradient (0 to 100% of 20 mM sodium acetate buffer,pH 5.5) in 10 CV. The purest CDH fractions were pooled, desalted with 20mM sodium acetate buffer, pH 5.5, concentrated and frozen at −70° C. forfurther use.

Example 7 Obtaining Nucleotide and Protein Sequences of New CDHs

Mycelium for nucleic acid isolations was harvested from celluloseinduced growing cultures after 5 days. The mycelium was frozen in liquidnitrogen and homogenized using mortar and pestle. Portions of 100 mgmycelium were used for DNA extraction (Liu et al., 2000, Journal ofClinical Microbiology, 38: 471). Total RNA was isolated using TriFast(Peqlab, Erlangen, Germany). cDNA synthesis was performed with the FirstStrand cDNA Synthesis Kit (Fermentas, Vilnius, Lithuania) and the anchorprimer (5′-GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTT-3′). Degenerated primer onthe basis of known ascomycete CDH sequences were used to amplifyfragments of genomic DNA encoding for CDH. For the amplification of theadjacent upstream region the DNA Walking SpeedUp Premix Kit (Seegene,Seoul, Korea) was used. For the amplification of the 3′ region cDNA wasused as a template. To obtain full-length cDNA clones encoding the CDHproteins a nested PCR with two specific forward primer upstream of theputative start codon and two reverse primer, one specific for a sequenceshortly downstream of the stop codon and the universal primer(5′-GTACTAGTCGACGCGTGGCC-3′) complementary to the anchor primer, wasdone. Names in the following primer table are abbreviated as follows:Chaetomium atrobrunneum, CA; Corynascus thermophilus, CT; Hypoxylonhaematostroma, HH; Neurospora crassa, NC; Stachybotrys bisbyi, SB.

forward primer reverse primer 5′-HH-1 atgcctctcttgtttggaccg Universal5′-HH-2 tcaactctcatacttggcttgg 3′-HH-1 TACATCCAGCTTACCGGCACTG 5′-CA-1TAGAGTCGAGGCGAACCAG UNIVERSAL 5′-CA-2 TTGCTGCTGTGCTCCTATGC 3′-CA-1ttccttccctccatcaactcc 5′-SB-1 tcttgctacgcacttcggtattg Universal 5′-SB-2TGTGTACCCTGTTTACTCACC 3′-SB-1 GTACCCATTAAGTACACTGCCAG 5′-CT-1TCTTATAAGCCTTTGGCTCC Universal 5′-CT-2 TTGGCTCCGTTGGAACAATG 3′-CT-1TTCCCCCTTCGAATTCGGTC 5′-NC-1 cgcaccaaccgtgtgaagtg Universal 5′-NC-2TACAAGATGAGGACCACCTCG 3′-NC-1 AGCTACCTATCACCCTCTGTC

The obtained PCR products were then fully sequenced to obtain thecomplete nucleic acid sequence of the respective cdh gene.

Example 8 Generation of Myriococcum thermophilum CDH Variants bySite-Directed Mutagenesis

For enhanced production of recombinant Myriococcum thermophilum CDH(Zamocky et al., 2008,) in Pichia pastoris the gene (gene bank accessioncode EF 492052, GI:164597963) was codon optimised (FIG. 1) forexpression in P. pastoris and synthesized by GenScript (Piscataway,N.J., USA). The gene shows a maximum similarity with CDH from Thielaviaheterothallica (74% identity) and only 63% identity with the gene fromHumicola insolens. On the protein level, the similarity is highest toThielavia heterothallica CDH (93% identity, 97% positives, 0% gaps) andquite low for Humicola insolens CDH (61% identity, 71% positives, 2%gaps).

The synthetic M. thermophilum CDH gene was mutated by a two-stepsite-directed mutagenesis protocol using PCR and Dpn/digestion. Theyeast vector pPICZ A carrying the synthetic CDH gene was used astemplate for mutagenic PCR. For the replacement of Asp160 with Lys theprimers 5′-TCCAAGCTTTTAAAGATCCAGGTAAC-3′ (Mt CDH-D181K-fw) and5′-AAAAGCTTGGACCCAACCAAG-3′ (Mt CDH-D181Krv) were used. For the doublemutant D547S/E550S primers 5′-GTCTTCTATTCTTTTTACTCTGCTTGGGATG-3′ (MtCDH-D547S/E550S-fw) and 5′-ATAGAAGACCACATCAGGG-3′ (MtCDH-D547S/E550S-rv) were used. The mutation sites are indicated by boldletters in the mutagenic forward primers. PCR was performed under thefollowing conditions: 98° C. for 30 s, then 32 cycles of 98° C. for 10s; 62° C. for 20 s; 72° C. for 2 min, with a 10 min final extension at72° C. The 50 μl reaction mix contained Phusion HF Buffer (New EnglandBiolabs, Ipswich, Mass., USA), 0.1 μg of plasmid DNA, 1 unit of PhusionDNA polymerase (New England Biolabs), 10 μM of each dNTP and 5 pmol ofeach primer.

PCR reactions were separated by agarose gel electrophoresis and bands at6 kB purified using the Wizard SV Gel and PCRCleanUp System (Promega,Madison, Wis., USA). The purified PCR fragment was digested with DpnI(Fermentas, Vilnius, Lithuania) to remove methylated DNA. 10 μl of thisreaction was used to transform chemically competent NEB-5-Alpha E. colicells (New England Biolabs) according to the manufacturer. For eachmutation 3 colonies were checked by sequencing for the presence of thecorrect mutation. The purified plasmid of a positive clone waslinearized with Sad and used to transform competent X-33 P. pastoriscells. Colonies growing on YPD zeocin agar plates (100 mg/L) werechecked by PCR for the integration of the construct. Two positive clonesof each mutation were further cultivated under induced condition andanalysed for CDH production. The clones with the highest yield wereselected for fermentation.

Example 9 Production of Recombinant CDH

An overnight pre-culture of a Pichia pastoris transformant (selectedfrom a YPD plate with 100 mg/L Zeocin) was inoculated into 0.3 L ofproduction stage medium in a Infors HT multifermenter (Bottmingen,Switzerland). The production stage medium contained per litre: 26.7 mlof H₃PO₄ (85%); 0.93 g of CaSO₄.2H₂O; 14.9 g of MgSO₄.7H₂O; 18.2 g ofK₂SO₄; 4.13 g of KOH; 4% (v/v) glycerol; 1.45 ml of PTM₁ trace elementsolution for P. pastoris according to the Invitrogen manual 053002 Ver.B (Carlsbad, Calif., USA). The PTM₁ trace element solution contains perlitre: 6 g of CuSO₄.5H₂O, 0.08 g of NaI, 3 g of MnSO₄.H₂O, 0.2 g ofNaMoO₄.2H₂O, 0.02 g of H₃BO₃, 0.5 g of CoCl₂, 20 g of ZnCl₂, FeSO₄7H₂O,0.2 g of biotin, 5 ml of sulfuric acid. A glycerol feed was performedwith an addition of 9 gL⁻¹h⁻¹ until the wet cell weight exceeded 150 gper litre. As soon as the residual glycerol was used up (determined bymonitoring the increase of the dissolved oxygen tension), a methanolfeed (100% methanol containing 12 ml PTM₁ trace element solution perlitre) with an average addition of 3 gL⁻¹h⁻¹ was started and continuedfor 72 h at 30° C. and 20% oxygen tension. The culture supernatant wasseparated from residual biomass by centrifugation (20 min, 6000×g) andconcentrated and purified by hydrophobic interaction chromatography. Tothat purpose, saturated ammonium sulphate solution was slowly added tothe clear culture supernatant at 4° C. to 20% final saturation. After asecond centrifugation step (30 min, 30,000×g) the solution was appliedto a PHE-Source column (GE Healthcare Biosciences) equilibrated with 100mM sodium acetate buffer, pH 5.5 containing (NH₄)₂SO₄ (20% saturation)and 0.2 M NaCl. The column was eluted with a linear gradient (0 to 100%of 20 mM sodium acetate buffer, pH 5.5) in 10 CV. The purest CDHfractions were pooled, desalted with 20 mM sodium acetate buffer, pH5.5, concentrated and stored for further use.

Example 10 Electrochemical Equipment

A three electrode flow through amperometric wall jet cell was used(Appelqvist et al, Anal. Chim. Acta, 169 (1985) 237-47.) and containedthe working electrode (graphite electrode modified with CDH), areference electrode (Ag|AgCl in 0.1 M KCl) and a counter electrode madeof a platinum wire, connected to a potentiostat (Zata Elektronik, Hoor,Sweden). The enzyme modified electrode was pressfitted into a Teflonholder and inserted into the wall jet cell and kept at a constantdistance (ca. 1 mm) from the inlet nozzle. The response currents wererecorded on a strip chart recorder (Kipp & Zonen, Delft, TheNetherlands). The electrochemical cell was connected on-line to a singleline flow injection (FI) system, in which the carrier flow wasmaintained at a constant flow rate of 0.5 ml min⁻¹ by a peristaltic pump(Gilson, Villier-1e-Bel, France). The injector was an electricallycontrolled six-port valve (Rheodyne, Cotati, Calif., USA), and theinjection loop volume was 50 μl.

For the screen-printed electrodes a special methacrylate wall jet flowfor flow injection analysis (FIA) from propSense (Oviedo, Spain) wasused. The electrochemical cell consists of a carbon working electrode (4mm diameter), a carbon counter electrode and silver reference electrodeconnected to a potentiostat (Zäta Elektronic). The response currentswere recorded on a strip chart recorder (Kipp & Zonen). Theelectrochemical cell was connected on-line to a single flow injection(FI) system, in which the carrier flow was maintained at a constant flowrate of 0.5 ml min⁻¹ by a peristaltic pump (Gilson). For injection anelectronically controlled six-port valve (Rheodyne) and a injection loop(50 μl) was used.

Example 11 Preparation of Enzyme Modified Graphite Electrodes

CDH was immobilised through simple chemo-physical adsorption onto thesurface of solid spectroscopic graphite electrodes (diameter=3.05 mm,Ringsdorff Spektralkohlestabe, SGL Carbon Sigri Greatlakes Carbon GroupRingsdorff-Werke GmbH, Bonn Germany). The electrode was cut and polishedon wet emery paper (Tufbak, Durite, P400) and afterwards carefullyrinsed with Milli-Q water and dried. Then 5 μl of enzyme solution wasspread onto the entire active surface of the electrode (0.0731 cm²). Theelectrode was dried at room temperature and then stored overnight at 4°C. Before use, the electrode was thoroughly rinsed with Milli-Q water inorder to remove any weakly adsorbed enzyme and plugged into in the walljet cell already containing buffer. Then, the required potential wasapplied until a stable background current was obtained before anysubstrate was injected into the flow system.

Example 12 Preparation of Enzyme-Modified Screen Printed Electrodes

Five μl of enzyme solution was placed on the carbon-based electrode(DropSens, Oviedo, Spain) so that the whole area was entirely coatedwith solution. The immobilisation was allowed to proceed overnight at 4°C. Before use the electrodes were thoroughly rinsed with water.Cross-linking of the biocomponent was carried out by chemicalmodification with glutaraldehyde where 1 μl of an aqueous 1%glutaraldehyde solution was applied on the enzyme layer at 37° C. for10-15 min. After rinsing the electrodes were allowed to dry at roomtemperature.

The optimum for the applied potential was determined with a 10 mMlactose solution. The potential was varied stepwise from −250 to +600 mVvs. Ag|AgCl in 0.1 M KCl and +300 mV chosen for further experiments.

Example 13 pH Profiles of CDH Immobilised on Electrodes

The activity versus pH-profile for direct electron transfer (DET) of theadsorbed enzyme was determined electrochemically using a flow injectionsystem. The substrate was lactose with a concentration of 5 mM. Asenzyme assays should proceed under saturating substrate conditions sothat slight variations in the absolute concentration have no influenceon the reaction rate an amount at least 10 times the K_(M)-value shouldbe present. The following buffers were used in the experiments: 50 mMsodium citrate buffer (pH 3.0-6.5), 50 mM sodium phosphate buffer (pH6.0-9.0). The buffers were degassed before use to prevent micro bubblesin the flow system.

Example 14 Heterogeneous Enzyme Kinetics on Electrodes

The kinetic parameters K_(M) (Michaelis-Menten constant) and v_(max)(maximum volumetric activity), in this case equal to I_(max) (maximumresponse in current), were determined for a number of substrates in theDET mode (the electron acceptor being the graphite electrode). Allkinetic parameters were calculated by nonlinear least-square regression,fitting the observed data to the Henri-Michaelis-Menten equation. Thesecalculations were done after correcting the substrate concentrationvalues using the dispersion factor of the flow system used including thewall jet cell by dividing the steady state current registered for a 50mM ferrocyanide solution with that of the peak current for the injectedsample having an equal concentration of ferrocyanide and using anapplied potential of 400 mV (Ruzicka and Hansen, Flow InjectionAnalysis, 2nd ed., Wiley, New York 1988). In our case, for a 1 mmdistance between electrode and inlet nozzle and 0.5 ml min⁻¹ flow rate,the dispersion factor D was equal to 1.18 (FIG. 5).

Example 15 CDH of Chaetomium atrobrunneum

A cellobiose dehydrogenase with high glucose turnover rates and activityunder physiological pH conditions was obtained from liquid cultures ofChaetomium atrobrunneum. The culture was grown and screened asdescribed. The maximum activity under the chosen conditions was 90 U/L(cyt c assay, pH 6.0, 11^(th) day). For enzyme production andpurification the outlined procedures were applied and resulted in an CDHpreparation with a specific activity of 11.7 U/mg (DCIP assay, pH 6.0),an apparent molecular weight of 90 kDa as determined by SDS-PAGE and anisoelectric point of 4.6. The calculated molecular weight of theobtained protein sequence is 86.047 kDa and fits well to the native CDH.The calculated isoelectric point is 5.0. The spectrum of Chaetomiumatrobrunneum CDH is typical and shows the haem alpha-, beta- andgamma-bands of the reduced enzyme at 563, 533 and 430 nm. In theoxidised enzyme the gamma-band has its absorption maximum at 421 nm witha shoulder at 450 nm, which disappears after reduction with lactose andcorresponds to the absorption peak of the FAD cofactor. Kineticcharacterisation with the cyt c assay and lactose as electron donorresulted in a neutral pH profile with an activity maximum at pH 5.0 andstill 18% relative activity at pH 7.4 (FIG. 2.a). The specific activityat pH 7.4 was 0.88 U/mg using the cyt c assay and glucose as substrate.The pH optimum of the flavin domain was obtained with the DCIP assay andshows a more acidic pH optimum, however, the flavin domain issufficiently active at pH 7.4 also with this electron acceptor. Kineticconstants for glucose (obtained with the cyt c assay at pH 5.0) are aK_(M) of 240 mM and a k_(cat) of 17.5 s⁻¹ for glucose, which shows incomparison to currently known enzymes a far better suitability of thisenzyme for the proposed application.

To test the electrochemical behaviour of Chaetomium atrobrunneum CDH onelectrodes, the purified enzyme preparation was immobilised byadsorption on a spectroscopic graphite electrode surface. Using a flowcell and subsequent injections of 50 mM glucose, DET currents weredetermined to determine the pH optimum, the current at the pH optimumand the current at pH 7.4. The optimum pH under the chosen conditions is5.6 and 48% of the maximum current was obtained at pH 7.4 in 10 mMphosphate buffered saline (PBS) containing 100 mM NaCl. The K_(M) valueof the heterogenised enzyme at optimum pH on the electrode surface wasdetermined to be 80 mM and I_(max)=30 nA. The currents obtained inglucose measurements should therefore follow a nearly linearrelationship for concentrations approx. five-fold below the K_(M) value.The DET current density obtained at pH 7.4 was 233 nA/cm² and the linearrange for glucose detection at pH 7.4 with the chosen setup within 3-15mM.

Example 16 CDH from Corynascus thermophilus

A cellobiose dehydrogenase with high glucose turnover rates and activityunder physiological pH conditions was obtained from liquid cultures ofCorynascus thermophilus. The maximum activity obtained was 1400 U/L (cytc assay, pH 6.0, 6^(th) day). For enzyme production and purification theoutlined procedures were applied and resulted in a CDH preparation witha specific activity of 17.9 U/mg, an apparent molecular weight of 87 kDaas determined by SDS-PAGE and an isoelectric point of 4.1. Thecalculated molecular weight of the obtained protein sequence is 81.946kDa and fits well to the native CDH. The calculated isoelectric point is4.64. The spectrum of C. thermophilus CDH is typical and shows the haemα-, β- and γ-bands of the reduced enzyme at 562, 533 and 429 nm. In theoxidised enzyme the γ-band has its absorption maximum at 420 nm with ashoulder at 450 nm, which disappears after reduction with lactose andcorresponds to the absorption peak of the FAD cofactor. Kineticcharacterisation with the cyt c assay resulted in a pH profile with anactivity maximum at pH 7.5 and 98% relative activity at pH 7.4 (FIG.2.b). The specific activity at pH 7.4 was 3.6 U/mg using the cyt c assayand glucose as substrate. The pH optimum of the flavin domain wasobtained with the DCIP assay and shows a more acidic pH optimum,however, the flavin domain is sufficiently active at pH 7.4 also withthis electron acceptor. Kinetic constants for glucose (obtained with thecyt c assay at pH 5.0) are a K_(M) of 950 mM and a k_(cat) of 32 s⁻¹ forglucose.

To test the electrochemical behaviour of C. thermophilus CDH onelectrodes, the purified enzyme preparation was immobilised byadsorption on a spectroscopic graphite electrode surface. Using a flowcell and subsequent injections of 50 mM glucose, DET currents weredetermined to determine the pH optimum, the current at the pH optimumand the current at pH 7.4. The optimum pH under the chosen conditions is8.5 and 96% of the maximum current was obtained at pH 7.4 in 10 mMphosphate buffered saline (PBS) containing 100 mM NaCl. The K_(M) valueof the heterogenised enzyme at optimum pH on the electrode surface wasdetermined to be 188 mM and I_(max)=190 nA. The currents obtained inglucose measurements should therefore follow a nearly linearrelationship for concentrations approx. five-fold below the K_(M) value.The DET current density obtained at pH 7.4 was 3500 nA/cm² and thelinear range for glucose detection at pH 7.4 with the chosen setupwithin 1-15 mM.

Example 17 CDH of Hypoxylon haematostroma

A cellobiose dehydrogenase with high glucose turnover rates and activityunder physiological pH conditions was obtained from liquid cultures ofHypoxylon haematostroma. The culture was grown and screened asdescribed. The maximum activity under the chosen conditions was 65 U/L(cyt c assay, pH 6.0, 9^(th) day). For enzyme production andpurification the outlined procedures were applied and resulted in an CDHpreparation with a specific activity of 15.3 U/mg (DCIP assay, pH 6.0),an apparent molecular weight of 85 Da as determined by SDS-PAGE and anisoelectric point of 4.1. The calculated molecular weight of theobtained protein sequence is 87.514 kDa and fits well to the native CDH.The calculated isoelectric point is 6.37. The spectrum of Hypoxylonhaematostroma CDH is typical and shows the haem alpha-, beta- andgamma-bands of the reduced enzyme at 563, 533 and 429 nm. In theoxidised enzyme the gamma-band has its absorption maximum at 421 nm witha shoulder at 450 nm, which disappears after reduction with lactose andcorresponds to the absorption peak of the FAD cofactor. Kineticcharacterisation with the cyt c assay and lactose as electron donorresulted in a neutral pH profile with an activity maximum at pH 5.5 andstill 65% relative activity at pH 7.4 (FIG. 2.c). The specific activityat pH 7.4 was 2.73 U/mg using the cyt c assay and glucose as substrate.The pH optimum of the flavin domain was obtained with the DCIP assay andshows a more acidic pH optimum, however, the flavin domain issufficiently active at pH 7.4 also with this electron acceptor. Kineticconstants for glucose (obtained with the cyt c assay at pH 5.5) are aK_(M) of 260 mM and a k_(cat) of 8.8 s⁻¹ for glucose, which shows incomparison to currently known enzymes a far better suitability of thisenzyme for the proposed application.

To test the electrochemical behaviour of Hypoxylon haematostroma CDH onelectrodes, the purified enzyme preparation was immobilised byadsorption on a spectroscopic graphite electrode surface. Using a flowcell and subsequent injections of 50 mM glucose, DET currents weredetermined to determine the pH optimum, the current at the pH optimumand the current at pH 7.4. The optimum pH under the chosen conditions is7.5 and the maximum current was obtained at this pH and at pH 7.4 in 10mM phosphate buffered saline (PBS) containing 100 mM NaCl. The K_(M)value of the heterogenised enzyme at optimum pH (7.4) on the electrodesurface for glucose was determined to be 49 mM and I_(max)=55 nA. Thecurrents obtained in glucose measurements should therefore follow anearly linear relationship for concentrations approx. five-fold belowthe K_(M) value. The DET current density obtained at pH 7.4 was 383nA/cm² and the linear range for glucose detection at pH 7.4 with thechosen setup within 2-20 mM.

Example 18 CDH of Neurospora crassa

A cellobiose dehydrogenase with high glucose turnover rates and activityunder physiological pH conditions was obtained from liquid cultures ofNeurospora crassa. The culture was grown and screened as described. Themaximum activity under the chosen conditions was 156 U/L (cyt c assay,pH 6.0, 18th day). For enzyme production and purification the outlinedprocedures were applied and resulted in an CDH preparation with aspecific activity of 10.6 U/mg (DCIP assay, pH 6.0), an apparentmolecular weight of 90 kDa as determined by SDS-PAGE and an isoelectricpoint of 4.3. The calculated molecular weight of the obtained proteinsequence is 86.283 kDa and fits well to the native CDH. The calculatedisoelectric point is 6.68. The spectrum of Neurospora crassa CDH istypical and shows the haem alpha-, beta- and gamma-bands of the reducedenzyme at 563, 533 and 430 nm. In the oxidised enzyme the gamma-band hasits absorption maximum at 421 nm with a shoulder at 450 nm, whichdisappears after reduction with lactose and corresponds to theabsorption peak of the FAD cofactor. Kinetic characterisation with thecyt c assay and lactose as electron donor resulted in a neutral pHprofile with an activity maximum at pH 6.0 and 52% relative activity atpH 7.4 (FIG. 2.d). The specific activity at pH 7.4 was 1.04 U/mg usingthe cyt c assay and glucose as substrate. The pH optimum of the flavindomain was obtained with the DCIP assay and shows a more acidic pHoptimum, however, the flavin domain is sufficiently active at pH 7.4also with this electron acceptor. Kinetic constants for glucose(obtained with the cyt c assay at pH 5.5) are a K_(M) of 1680 mM and akcat of 15.9 for glucose, which shows in comparison to other knownenzymes a far better suitability of this enzyme for the proposedapplication.

To test the electrochemical behaviour of Neurospora crassa CDH onelectrodes, the purified enzyme preparation was immobilised byadsorption on a spectroscopic graphite electrode surface. Using a flowcell and subsequent injections of 50 mM glucose, DET currents weredetermined to determine the pH optimum, the current at the pH optimumand the current at pH 7.4. The optimum pH under the chosen conditions is5.0 and 31% of the maximum current was obtained at pH 7.4 in 10 mMphosphate buffered saline (PBS) containing 100 mM NaCl. The K_(M) valueof the heterogenised enzyme at optimum pH on the electrode surface wasdetermined to be 90 mM and I_(max)=5 nA. The currents obtained inglucose measurements should therefore follow a nearly linearrelationship for concentrations approx. five-fold below the K_(M) value.The DET current density obtained at pH 7.4 was 82 nA/cm² and the linearrange for glucose detection at pH 7.4 with the chosen setup within 2-10mM.

Example 19 CDH of Stachybotris bisbyi

A cellobiose dehydrogenase with high glucose turnover rates and activityunder physiological pH conditions was obtained from liquid cultures ofStachybotris bisbyi. The culture was grown and screened as described.The maximum activity under the chosen conditions was 154 U/L (cyt cassay, pH 6.0, 24^(th) day). For enzyme production and purification theoutlined procedures were applied and resulted in a CDH preparation witha specific activity of 7.9 U/mg (DCIP assay, pH 6.0), an apparentmolecular weight of 100 kDa as determined by SDS-PAGE and an isoelectricpoint of 4.5. The calculated molecular weight of the obtained proteinsequence is 86.212 kDa and fits well to the native CDH when consideringa glycosylation of 14% of S. bisbyi CDH, a value which lies within theobserved range (2-15%, Zámocký et al., 2006, Current Protein and PeptideScience, 7: 255-280). The calculated isoelectric point is 6.37. Thespectrum of Stachybotris bisbyi CDH is typical and shows the haemalpha-, beta- and gamma-bands of the reduced enzyme at 562, 533 and 430nm. In the oxidised enzyme the gamma-band has its absorption maximum at420 nm with a shoulder at 450 nm, which disappears after reduction withlactose and corresponds to the absorption peak of the FAD cofactor.Kinetic characterisation with the cyt c assay resulted in a pH profilewith an activity maximum at pH 5.5 and 60% relative activity at pH 7.4(FIG. 2.e). The specific activity at pH 7.4 was 0.58 U/mg using the cytc assay and glucose as substrate. The pH optimum of the flavin domainwas obtained with the DCIP assay and shows a similar trend indicatingthat substrate oxidation by the enzyme is efficient at pH 7.4. Kineticconstants for glucose (obtained with the cyt c assay at pH 5.5) are aK_(M) of 950 mM and a k_(cat) of 14.1 s⁻² for glucose, which shows incomparison to currently known enzymes a far better suitability of thisenzyme for the proposed application.

To test the electrochemical behaviour of Stachybotris bisbyi CDH onelectrodes, the purified enzyme preparation was immobilised byadsorption on a spectroscopic graphite electrode surface. Using a flowcell and subsequent injections of 50 mM glucose, DET currents weredetermined to determine the pH optimum, the current at the pH optimumand the current at pH 7.4. The optimum pH under the chosen conditions is5.0 and 27% of the maximum current was obtained at pH 7.4 in 10 mMphosphate buffered saline (PBS) containing 100 mM NaCl. The K_(M) valueof the heterogenised enzyme at optimum pH on the electrode surface wasdetermined to be 131 mM and I_(max)=65 nA. The currents obtained inglucose measurements should therefore follow a nearly linearrelationship for concentrations approx. five-fold below the K_(M) value.The DET current density obtained at pH 7.4 was 237 nA/cm² and the linearrange for glucose detection at pH 7.4 with the chosen setup within 3-15mM.

Example 20 CDH from Myriococcum thermophilum

CDH from Myriococcum thermophilum was found to oxidise glucose veryefficiently (Harreither et al., 2007, Electroanalysis 19: 172-180), butnot under physiological conditions. It was used as a protein scaffoldfor which DET at neutral pH was developed by means of geneticengineering. The enzyme variants D181K and D547S/E550S were obtainedaccording to the described methods to increase the IET at pH 7.4 andthereby the electrode response in order to optimise the enzyme forapplications under neutral or alkaline pH conditions.

For enzyme production and purification of the enzyme from the nativeproducer, the protocol given in (Harreither et al., 2007,Electroanalysis 19: 172-180) was followed and resulted in a CDHpreparation with a specific activity of 10.7 U/mg (DCIP assay, pH 6.0),an apparent molecular weight of 94 kDa and an isoelectric point of 3.8.The calculated molecular weight of the protein sequence is 86.701 kDaand fits well to the native CDH. The calculated isoelectric point is4.62. The spectrum of CDH obtained from M. thermophilum is typical andshows the haem alpha-, beta- and gamma-bands of the reduced enzyme at563, 533 and 429 nm. In the oxidised enzyme the gamma-band has itsabsorption maximum at 421 nm with a shoulder at 450 nm, which disappearsafter reduction with lactose and corresponds to the absorption peak ofthe FAD cofactor. Kinetic characterisation with the cyt c assay andlactose as electron donor resulted in a neutral pH profile with anactivity maximum between pH 4.0-4.5 and 0% relative activity at pH 7.4(FIG. 2.f). The specific activity at pH 7.4 was also 0 U/mg using thecyt c assay and glucose as substrate. The pH optimum of the flavindomain was obtained with the DCIP assay and shows a far less acidic pHoptimum (6.0), indicating that substrate oxidation at the flavin domainis performed even at neutral and slightly alkaline conditionsefficiently, but the IET is rate limiting. The obtained kineticconstants for glucose (DCIP assay, pH 6.0; K_(M)=250 mM, kcat=14.2 s⁻¹)show that although glucose conversion is very efficient, M. thermophilumCDH is not suitable for the proposed application because no IET wasmeasured above pH 7.0.

The recombinant enzyme variants were produced heterologeously in P.pastoris according to the explained routines. The molecular weights,isoelectric points or spectral properties did not differ significantlyfrom the native enzyme produced by the fungus.

Kinetic characterisation of D181K with the cyt c assay and lactose aselectron donor resulted in a pH with an activity maximum at 5.0 and 24%relative activity at pH 7.4 (FIG. 2.g). The specific activity at pH 7.4was 1.01 U/mg using the cyt c assay and glucose as substrate. To testthe electro-chemical behaviour of D181K on electrodes, the purifiedenzyme preparation was immobilised by adsorption on a screen printedelectrode. Using a flow cell and subsequent injections of 50 mM glucose,DET currents were determined to determine the pH optimum, the current atthe pH optimum and the current at pH 7.4. The optimum pH under thechosen conditions is 5.5 and 52% of the maximum current was obtained atpH 7.4 in 10 mM phosphate buffered saline (PBS) containing 100 mM NaCl.The K_(M) value of the heterogenised enzyme at optimum pH on theelectrode surface was determined to be 133 mM and I_(max)=105 nA. Thecurrents obtained in glucose measurements should therefore follow anearly linear relationship for concentrations approx. five-fold belowthe K_(M) value. The DET current density obtained at pH 7.4 was 513nA/cm² and the linear range for glucose detection at pH 7.4 with thechosen setup within 0.5-20 mM.

Kinetic characterisation of D181R with the cyt c assay and lactose aselectron donor resulted in a pH with an activity maximum at 5.0 and 20%relative activity at pH 7.4. The specific activity at pH 7.4 was 0.73U/mg using the cyt c assay and glucose as substrate. To test theelectrochemical behaviour of D181R on electrodes, the purified enzymepreparation was immobilised by adsorption on a screen printed electrode.Using a flow cell and subsequent injections of 50 mM glucose, DETcurrents were determined for pH 5.0 and 7.4 in 10 mM phosphate bufferedsaline (PBS) containing 100 mM NaCl. The DET current density obtained atpH 5.0 and 7.4 was 485 nA/cm² and 168 nA/cm², respectively. Theelectrode had 42% of the maximum current at pH 7.4.

Kinetic characterisation of D198K with the cyt c assay and lactose aselectron donor resulted in a pH with an activity maximum at 5.0 and 22%relative activity at pH 7.4. The specific activity at pH 7.4 was 0.62U/mg using the cyt c assay and glucose as substrate. To test theelectrochemical behaviour of D198K on electrodes, the purified enzymepreparation was immobilised by adsorption on a screen printed electrode.Using a flow cell and subsequent injections of 50 mM glucose, DETcurrents were determined for pH 5.0 and 7.4 in 10 mM phosphate bufferedsaline (PBS) containing 100 mM NaCl. The DET current density obtained atpH 5.0 and 7.4 was 259 nA/cm² and 108 nA/cm², respectively. Theelectrode had 42% of the maximum current at pH 7.4.

Kinetic characterisation of D198N with the cyt c assay and lactose aselectron donor resulted in a pH with an activity maximum at 5.0 and 22%relative activity at pH 7.4. The specific activity at pH 7.4 was 0.69U/mg using the cyt c assay and glucose as substrate. To test theelectrochemical behaviour of D198N on electrodes, the purified enzymepreparation was immobilised by adsorption on a screen printed electrode.Using a flow cell and subsequent injections of 50 mM glucose, DETcurrents were determined for pH 5.0 and 7.4 in 10 mM phosphate bufferedsaline (PBS) containing 100 mM NaCl. The DET current density obtained atpH 5.0 and 7.4 was 353 nA/cm² and 140 nA/cm², respectively. Theelectrode had 40% of the maximum current at pH 7.4.

Kinetic characterisation of D568S/E571S with the cyt c assay and lactoseas electron donor resulted in a pH with an activity maximum between 4.5and 5.0 and 13% relative activity at pH 7.4 (FIG. 2.h). The specificactivity at pH 7.4 was 0.70 U/mg using the cyt c assay and glucose assubstrate. To test the electrochemical behaviour of D568S/E571S onelectrodes, the purified enzyme preparation was immobilised byadsorption on a graphite electrode surface. Using a flow cell andsubsequent injections of 50 mM glucose, DET currents were determined todetermine the pH optimum, the current at the pH optimum and the currentat pH 7.4. The optimum pH under the chosen conditions is 5.5 and 24% ofthe maximum current was obtained at pH 7.4 in 10 mM phosphate bufferedsaline (PBS) containing 100 mM NaCl. The K_(M) value of theheterogenised enzyme at optimum pH on the electrode surface wasdetermined to be 55 mM and I_(max)=30 nA. The currents obtained inglucose measurements should therefore follow a nearly linearrelationship for concentrations approx. five-fold below the K_(M) value.The DET current density obtained at pH 7.4 was 241 nA/cm² and the linearrange for glucose detection at pH 7.4 with the chosen setup within 1-20mM.

Kinetic characterisation of D568S/E571S/D574S with the cyt c assay andlactose as electron donor resulted in a pH with an activity maximum at5.5 and 43% relative activity at pH 7.4. The specific activity at pH 7.4was 2.49 U/mg using the cyt c assay and glucose as substrate. To testthe electrochemical behaviour of D568S/E571S/D574S on electrodes, thepurified enzyme preparation was immobilised by adsorption on a screenprinted electrode. Using a flow cell and subsequent injections of 50 mMglucose, DET currents were determined for pH 5.0 and 7.4 in 10 mMphosphate buffered saline (PBS) containing 100 mM NaCl. The DET currentdensity obtained at pH 5.0 and 7.4 was 455 nA/cm² and 184 nA/cm²,respectively. The electrode had 40% of the maximum current at pH 7.4.

Kinetic characterisation of E571K with the cyt c assay and lactose aselectron donor resulted in a pH with an activity maximum at 5.0 and 17%relative activity at pH 7.4. The specific activity at pH 7.4 was 0.50U/mg using the cyt c assay and glucose as substrate. To test theelectrochemical behaviour of E571K on electrodes, the purified enzymepreparation was immobilised by adsorption on a screen printed electrode.Using a flow cell and subsequent injections of 50 mM glucose, DETcurrents were determined for pH 5.0 and 7.4 in 10 mM phosphate bufferedsaline (PBS) containing 100 mM NaCl. The DET current density obtained atpH 5.0 and 7.4 was 595 nA/cm² and 197 nA/cm², respectively. Theelectrode had 33% of the maximum current at pH 7.4.

Comparative Example 21 CDH from Myriococcum thermophilum—pH Profile ofGlucose with Wild-Type Enzyme using Graphite Electrodes

CDH from Myriococcum thermophilum was found to oxidise manycarbohydrates, glucose being one of them (Harreither et al., 2007,Electroanalysis 19: 172-180). From a pH profile measured with 5 mMcellobiose or lactose (FIG. 3A Harreither et al. 2007) a DET current atpH 7.5 can be seen with approx. 17% and 20%, respectively, of the valueof peak maximum at pH 5. One could speculate that glucose could also bedetected by this method, therefore a comparative measurement wasperformed using the same experimental conditions, enzyme and electrodepreparation procedures (50 mM sodium citrate buffer, pH 4.0, 4.5, 5.0,5.5, 6.5, 7.5; potential 400 mV vs. Ag|AgCl in 0.1 M KCl), exchangingthe originally used 5 mM cellobiose or 5 mM lactose for 5 mM glucose.The results are given in FIG. 2.i and show a strong decrease of thedetected current already at pH 6.5. At pH 7.5 no signal could bedetected and therefore no value calculated as the response was withinthe electronic noise of the measurement (2 nA). The reason for thisbehaviour lies in the higher K_(M) value of M. thermophilum CDH forglucose (K_(M)=240 mM) than for cellobiose (K_(M)=0.027 mM) or lactose(K_(M)=0.055 mM, all data from Harreither et al. 2007) which reduces theobtained current at pH 7.5 below the limit of detection.

Example 22 Sequences

>M.thermophilum (SEQ ID NO: 1)mrtssrligalaaallpsalagnnvpntftdpdsgitfntwgldedspqtqggftfgvalpsdalttdasefigylkcarndesgwcgislggpmtnsllitawphedtvytslrfatgyampdvyegdaeitqvsssvnsthfslifrcknclqwshggssggastsggvlvlgwvqafddpgnptcpeqitlqqhdngmgiwgaqlntdaaspsytdwaaqatktvtgdcegptetsvvgvpvptgvsfdyivvgggaggipaadklseagksvlliekgfastantggtlgpewleghdltrfdvpglcnqiwvdskgiacedtdqmagcvlgggtavnaglwfkpysldwdylfpdgwkyndvqpainralsripgtdapstdgkryyqegfevlskglaaggwtsvtannapdkknrtfahapfmfaggerngplgtyfqtakkrnnfdvwlntsvkrviregghitgvevepfrdggyegivpvtkvtgrvilsagtfgsakillrsgigpedqlevvaasekdgptmignsswinlpvgynlddhlntdtvishpdvvfydfyeawddpiesdknsylesrtgilaqaapnigpmfweeivgadgivrqlqwtarvegslgapnghtmtmsqylgrgatsrgrmtitpslttivsdvpylkdpndkeaviqgiinlqnalqnvanltwlfpnstitpreyvesmvvspsnrrsnhwmgtnklgtddgrkggsavvdldtrvygtdnlfvidasifpgvpttnptsyivvaaehassrilalpdlepvpkygqcggrewtgsfvcadgstceyqnewysqcl >H.haematostroma (SEQ ID NO: 3)mgrlgslaklllavglnvqqcfgqngpptpytdsetgitfatwsggnglapwggltfgvalpenalttdateligylkcgsngtttdawcglsfggpmtnslllmawphedeiltsfrfasgytrpdlytgdakltqisstidkdhftlifrcqnclawnqdgasgsastsagslilgwasalraptnagcpaeinfnfhnngqmiwgatldesaanpsysewaakatatvtgdcggatpttttttttsvptatgipvptgtydyivvgagaggipladklseagksvlliekgppssgrwggtlkpewlkdtnltrfdvpglcnqiwvnsagvactdtdqmagcvlgggtavnaglwwkpynldwdynfprgwksrdmaaatrrvfsripgtdnpsmdgkrylqqgfeilagglkaagwtevtandapnkknhtyshspfmfsggerggpmgtylvsasrrknfhlwtgtavkrvvrtgghitglevepfvnggytgvvnvtsitgrvvlsagafgsakillrsgigpedqleivksstdgptmisdsswitlpvgynledhtntdtvvthpdvvfydfyeaghpnvtdkdlylnsragilaqaapnigpmfweeikgrdgvvrqlqwtarvegsagtpngyamtmsqylgrgaksrgrmtitkalttvvstvpylqdkndveaviqgiknlqaalsnvknltwayppsnttvedfvnnmlvsytnrrsnhwigtnklgtddgrsrggsavvdlntkvygtdnlfvvdagifpghittnptsyiviaaeraserildlpparaqprfaqcggrtwtgsfqcaapytcqyrnerysqcr >C.attrobruneum (SEQ ID NO: 5)mrpssrfvgalaaaasflpsalaqnnaavtftdpdtgivfnswglangapqtqggftfgvalpsdalttdatefigylecasadnqgwcgvsmggpmtnsllitawphednvytslrfatgyampdvysgdatitqisssinathfklifrcqnclqwthdgasggastsagvlvlgwvqafpspgnptcpdqitleqhnngmgiwgavmdsnvanpsytewaaqatktveaecdgpsetdivgvpvptgttfdyivvgggaggiptadklseagksvlliekgiastaehggtlgpewlegndltrfdvpglcnqiwvdskgiacedtdqmagcvlgggtavnaglwfkpysldwdylfpsgwkyrdiqaaigrvfsripgtdapstdgkryyqqgfdvlagglsaggwnkvtansspdkknrtfsnapfmfsggerggplatyltsakkrsnfnlwlntsvkrviregghvtgvevepfrtggyqgivnvtaysgrvvlsagtfgsakillrggigpadqlevvkaskidgptmisnaswiplpvgynlddhlntdtvithpdvafydfyeawntpieadknsylssrtgilaqaapnigpmmweeikgadgivrqlqwtarvegsfdtpngqamtisqylgrgatergrmtitpslttvvedvpylkdpndkeaviqgivnlqnalknvagltwtypnssitpreyvdnmvvspsnrranhwmgtakigtddgrlaggsavvdlntkvygtdnlfvvdasifpgtpttnpsayivtaaehasqrilglaapkpvgkwgqcggrqwtgsfqcvsgtkcevvnewysqcl >C.thermophilum (SEQ ID NO: 7)mkllsrvgatalaatlslkqcaaqmtegtytheatgitfktwtpsdgstftfglalpgdaltndateyigllrcqitdpsspgycgishgqsgqmtqalllvawasedvvytsfryatgytlpelytgdakltqiassysgdsfevlfrcencfswdqngatgsvstsngalvlgyaasksgltgatcpdtaefgfhnngfgqwgavlegatsdsyeewaqlatitppttcdgngpgdkvcvpapedtydylvvgagaggitvadklseaghkvlliekgppstglwngtmkpewlegtdltrfdvpglcnqiwydsagiactdtdqmagcvlgggtavnaglwwkphpadwddnfphgwkssdladatervfsripgtwhpsqdgklyrqegfevisqglanagwrevdanqepseknrtyshsvfmfsggerggplatylasaaqrsnfnlwvntsvrrairtgprvsgvelecladggfngtvnlkegggvifsagafgsaklllrsgigpedqleivasskdgetfiskndwiklpvghnlidhlntdliithpdvvfydfyaawdnpitedkeaylnsrsgilaqaapnigplmweevtpsdgitrqfqwtcrvegdssktnsthamtlsqylgrgvvsrgrmgitsgltttvaehpylhndgdleaviqgiqnvvdalsqvpdlewvlpppnttveeyvnslivspanrranhwmgtakmglddgrsggsavvdlntkvygtdnlfvvdasifpgmstgnpsamivivaeqaaqrilslry >S.bisbyi (SEQ ID NO: 9)mlfklsnwllalalfvgnvvaqlegptpytdpdtgivfqswvnpagtlkfgytypanaatvaatefigflecqgagwcsvslggsmlnkplvvaypsgdevlaslkwatgyanpepyggnhklsqisssvtsagfrvvyrcegclawnyqgieggsptngasmpigwaysassvlngdcvdntvliqhdtfgnygfvpdesslrteyndwtelptrvvrgdcggstttssvpsstappqgtgipvptgasydyivvgsgaggipiadklteagkkvlliekgppssgrydgklkptwlegtnltrfdvpglcnqiwvdsagiacrdtdqmagcvlgggtavnaglwwkpnpidwdynfpsgwkssemigatnrvfsriggttvpsqdgktyyqqgfnvlssglkaagwtsvslnnapaqrnrtygagpfmfsggerggplatylatakkrgnfdlwtntqvkrvirqgghvtgvevenyngdgykgtvkvtpvsgrvvlsagtfgsaklllrsgigpkdqlaivknstdgptmaserdwinlpvgynledhtntdivishpdvvhydfyeawtasiesdktaylgkrsgilaqaapnigplffdevrgadnivrsiqytarvegnsvvpngkamvisqylgrgavsrgrmtisqglntivstapylsnvndleaviksleniansltskvknlkiewpasgtsirdhytnmpldpatrranhwigtnkigtkngrltggdsvvdlntkvygtdnlfvvdasifpgmvttnpsayiviaaehaaskilslptakaaakyeqcggleyngnfqcasgltctwlndyywqct >N.crassa (SEQ ID NO: 11)MRTTSAFLSGLAAVASLLSPAFAQTAPKTFTHPDTGIVFNTWSASDSQTKGGFTVGMALPSNALTTDATEFIGYLECSSAKNGANSGWCGVSLRGAMTNNLLITAWPSDGEVYTNLMFATGYAMPKNYAGDAKITQIASSVNATHFTLVFRCQNCLSWDQDGVTGGISTSNKGAQLGWVQAFPSPGNPTCPTQITLSQHDNGMGQWGAAFDSNIANPSYTAWAAKATKTVTGTCSGPVTTSIAATPVPTGVSFDYIVVGGGAGGIPVADKLSESGKSVLLIEKGFASTGEHGGTLKPEWLNNTSLTRFDVPGLCNQIWKDSDGIACSDTDQMAGCVLGGGTAINAGLWYKPYTKDWDYLFPSGWKGSDIAGATSRALSRIPGTTTPSQDGKRYLQQGFEVLANGLKASGWKEVDSLKDSEQKNRTFSHTSYMYINGERGGPLATYLVSAKKRSNFKLWLNTAVKRVIREGGHITGVEVEAFRNGGYSGIIPVTNTTGRVVLSAGTFGSAKILLRSGIGPKDQLEVVKASADGPTMVSNSSWIDLPVGHNLVDHTNTDTVIQHNNVTFYDFYKAWDNPNTTDMNLYLNGRSGIFAQAAPNIGPLFWEEITGADGIVRQLHWTARVEGSFETPDGYAMTMSQYLGRGATSRGRMTLSPTLNTVVSDLPYLKDPNDKAAVVQGIVNLQKALANVKGLTWAYPSANQTAADFVDKQPVTYQSRRSNHWMGTNKMGTDDGRSGGTAVVDTNTRVYGTDNLYVVDASIFPGVPTTNPTAYIVVAAEHAAAKILAQPANEAVPKWGWCGGPTYTGSQTCQAPYKCEKQNDWYWQCV >M.thermophilum (SEQ ID NO: 2)atgaggacctcctctcgtttaatcggagcccttgcggcggcacttttgccgtctgcccttgcccagaacaatgtcccgaatacttttaccgaccctgactcgggcatcaccttcaacacgtggggtctcgacgaggattctccccagactcagggcggtttcaccttcggcgttgccctgccctctgatgccctcacaaccgacgcctcggaatttatcggttacttgaaatgcgcaaggaatgatgagagcggttggtgtggcatttcccttggcgggcctatgaccaactcgctcctcatcacagcctggccgcacgaggacacggtctacaccagtcttcggttcgcgaccggttacgccatgccggatgtctacgagggggacgccgagattacccaggtctcttcctctgttaattcgacgcacttcagtctcatcttcaggtgcaagaactgcctgcaatggagccacggcggctcctccggcggcgcctctacctcgggcggcgtgttggtactcggctgggttcaggcattcgacgatcccggcaatccaacctgccccgagcagatcacactccagcagcacgacaacggcatgggtatctggggtgcccagctcaacacggatgctgccagcccgtcctacactgactgggccgcccaggctaccaagaccgtcaccggtgactgcgagggccccaccgagacttctgtcgtcggcgtccccgttccgacgggtgtctcgttcgattatattgttgtcggcggcggcgccgggggcatccccgcagctgacaagctcagcgaggccggcaagagtgtgttgctcatcgagaagggctttgcttcgaccgcaaacaccggaggtactctcggccctgaatggcttgagggccatgatctgacccgcttcgacgtgccgggtctgtgcaaccagatctgggtcgattccaaggggatcgcttgcgaggataccgaccagatggctggctgtgttctcggcggcggcaccgccgtgaatgctggcctgtggttcaagccctactcgctcgactgggactacctcttccccgatggttggaagtacaatgacgtccagcctgccatcaaccgcgccctctcgcgcatcccaggcaccgacgccccttctaccgacggaaagcgctactaccaggagggttttgaggtcctctccaagggcctggccgccggcggctggacctcagtcacggccaataatgcgcccgacaagaagaaccgcaccttcgcccatgctcccttcatgtttgccggcggcgagcgcaatggccctctgggtacctacttccagactgccaagaagcgcaacaatttcgatgtctggctcaacacgtcggtcaagcgcgtcatccgtgagggtggccacatcaccggcgtcgaggtcgagccgttccgtgacggtggttacgagggcattgtccccgtcaccaaggttaccggccgcgttatcctgtctgccggcaccttcggcagtgcaaagattctgttaaggagcggtattggcccggaagatcagctagaagttgtcgcggcctccgagaaggacggccctaccatgatcggcaactcgtcctggatcaacctgcctgtcgggtacaacctcgatgaccatctcaacaccgacacagtcatctcccaccccgatgtcgtgttctacgacttttacgaggcgtgggatgatcccatcgagtctgacaagaatagctatctcgaatcgcgtacgggcatcctcgcccaagccgctcccaacattggccctatgttctgggaagagatcgtgggcgcggacggcatcgttcgccagctccagtggactgcccgtgtcgagggtagcctgggcgctcccaacggccacactatgaccatgtcgcagtaccttggccgtggtgccacctcacgcggccgcatgaccatcaccccgtctctgacgactatcgtctcagacgtgccttacctcaaagaccccaacgacaaggaggctgtcatccaaggcatcatcaacctgcagaacgcccttcagaacgtcgccaacctgacttggctcttccccaactctaccattacgccgcgcgaatacgttgagagcatggtcgtctccccgagcaaccggcggtccaaccactggatgggcaccaacaagctcggtaccgacgacgggcggaagggtggctccgctgtcgtcgacctcgacaccagggtctacggtactgacaacctcttcgtcatcgacgcctccatcttccccggcgtgcccaccacgaatcctacttcgtacatcgtggtagcggcagagcacgcttcgtcccgcatcctcgccctgcccgacctcgagcccgtccccaagtacggccagtgtggcggtcgcgaatggaccggtagcttcgtctgcgccgatggttccacgtgcgagtaccagaatgagtggtactcgcagtgcttgtga >H.haematostroma (SEQ ID NO: 4)atgggtcgcctaggctctctcgcgaagttgcttctcgcagtcggcttgaatgttcagcaatgcttcgggcaaaacggacccccgaccccctacactgatagtgagaccggtatcactttcgccacctggtccggcggaaacggcttagcaccctggggcggcttgactttcggtgttgcgttacctgaaaatgccctgaccaccgacgctaccgagctgattggatacctgaaatgcggttccaatggcacaaccacagatgcgtggtgtggtctgtcgtttgggggcccgatgactaacagcctccttctcatggcctggccgcacgaagacgagatcttgacatcattccgttttgccagtggatataccagaccagacctatacaccggcgatgccaaattaacgcagatatcatccaccatcgataaagatcactttactctaattttcaggtgccagaactgtctagcgtggaaccaagacggcgcgtctggttccgcttcaactagtgccggctccttgatattaggctgggccagtgcgcttcgggccccgacgaatgcaggctgtccggctgaaatcaacttcaacttccacaacaatggccagatgatatggggcgctacattagacgagagcgccgcaaacccatcatattcggaatgggctgccaaagccaccgctacggttaccggtgactgcggcggtgcaacccctacgaccactactaccaccaccacgtccgtccctaccgccacaggtatcccagtgccaactggcacctacgactatattgtagttggtgcgggtgctggcggaatacctttggccgacaagctgagcgaggctggaaagagtgtgttactgatcgaaaaggggccgccatcatcgggacgatggggtggcaccctcaagccagagtggttgaaggacaccaacttgacacggtttgacgtccctggcctgtgcaatcagatctgggtcaactctgcaggcgtcgcttgtactgacacagaccaaatggccggttgcgttcttggtggtggtacagctgtcaacgctggcctatggtggaagccctacaacctcgactgggattataacttcccacgcggatggaagtccagggatatggccgctgcaaccaggagagtcttctctcgcattcccggtacagataatccctcaatggatggcaagcggtatttacagcaaggcttcgaaatcctcgctggtggcttgaaagccgctggatggaccgaggttaccgcgaatgacgcacccaataagaagaaccacacctactcacactcgccgttcatgttctccggcggcgaacggggtggcccaatgggcacctacctggtatcggccagtagacgtaagaatttccatctatggacgggaacagcagtgaagagggttgttcgcacaggcggccatatcaccggtctggaggtcgagcccttcgtaaacggcggttataccggtgttgtcaacgtcacctcgattactggtcgggtcgtcttgtctgctggtgcgttcgggtcggctaagatattactgaggagcggcatcggacctgaggatcagttggagattgtcaagtcatcaaccgatggcccgaccatgatttccgattcttcttggattacgctacccgtcggttataatctagaggatcacacaaacaccgacacggtcgttacgcatcctgacgtcgtattttacgacttctacgaggctggacatcctaatgttaccgacaaggacttgtatctcaactcacgggccggaatccttgctcaagcagcgcctaatatcggcccaatgttctgggaagagattaagggtagggacggcgtcgttagacagctccagtggacagccagagttgaaggaagtgccggtacaccgaatgggtacgccatgacaatgagccaataccttggacgaggcgctaagtcgaggggccgaatgactatcacgaaggcgttgacgaccgtcgtttctacagtaccttacctacaggataagaacgacgtggaagcagtcatccagggaatcaagaaccttcaagcagcactttcgaacgtgaagaatctcacatgggcctacccaccatctaatacgacggtggaggactttgttaacaacatgctggtttcatacactaataggcgttccaaccactggattgggaccaacaagctcggaaccgatgatggccgatcgcgcggaggttcagctgtcgtggacctcaacactaaggtatacggcaccgacaacctgttcgtcgttgacgcaggaatattccccggtcatattaccacgaacccgacttcgtatatcgtgatcgccgctgagcgcgcttctgagaggatcctcgaccttcccccggctagagcacaaccgcgcttcgcgcagtgcggcgggcgaacgtggacgggtagcttccagtgtgcagcgccgtacacttgtcagtacaggaatgagc ggtattcccagtgccggtaa>C.attrobruneum (SEQ ID NO: 6)atgaggccctcctctcggtttgttggtgccctggcggcggcggcgtcgttcctgccgtctgcccttgcccagaacaatgctgcagtcaccttcactgacccggacaccggcatcgtcttcaactcctggggtcttgccaatggagcaccacagactcagggaggcttcacctttggtgtcgctctgccctctgatgcgctcacgaccgatgctaccgagttcattggttatttggaatgtgcctccgcggacaaccagggctggtgcggtgtctcgatgggcggccccatgaccaactcgcttcttatcaccgcctggccgcacgaggacaacgtctacacctccctccggtttgcaacaggatacgccatgccggatgtctactcgggagacgccaccatcacgcagatctcgtcgagcatcaacgcgacccacttcaagctcatcttcaggtgccagaactgcctgcaatggacccacgacggcgcttccggtggcgcctccacgtctgccggtgttctggtcctcggctgggtccaggctttcccttcccctggcaacccgacgtgcccggaccagatcacgctcgagcagcacaacaacggcatgggcatctggggtgcggtgatggactccaacgtcgccaacccgtcctacacagagtgggccgcgcaggccaccaagacggtcgaggccgagtgcgacggcccgagtgagacggatattgtcggcgtgcccgtgccgaccggcaccaccttcgactacatcgtcgtgggcggcggtgccggcggtatccccactgccgacaagctcagcgaggccggcaagagtgtgctgctgattgagaagggcatcgcctcgactgctgagcacggcggcactctcggacccgagtggctcgagggcaacgacctgacgcggttcgacgtgcccggtctttgcaaccagatctgggttgactccaagggcatcgcctgcgaggacaccgaccagatggccggttgcgtcctcggcggcggcacggccgtcaacgccggcctctggttcaagccctactcgctcgactgggactacctcttcccaagcggctggaagtaccgcgacatccaggccgccatcggcagggtgttctcgcgcatcccgggcactgacgcgccctcgaccgacggcaagcgctactaccagcagggcttcgacgtgctcgcgggcggcctgagtgccggcggctggaacaaggtcacggccaactcgtctccagacaagaagaaccgcaccttctcgaacgcgcctttcatgttctcgggcggcgagcgcggcgggcccctggccacttatctcaccagcgccaagaagcgcagcaacttcaacctgtggctcaacacgtcggtcaagcgcgtcatccgtgagggcggccacgtcacaggtgtcgaggtcgagcctttccggacgggcgggtaccagggtatcgtgaacgttaccgccgtttcgggccgtgtcgtcctgtcggctggtaccttcggcagtgccaagattctgctcagaggcggtattggcccagcggatcagctcgaggttgtcaaggcgtcgaagatcgacgggccgaccatgatcagcaatgcgtcttggattcctctgcctgttgggtacaacctggatgaccatctcaacactgacactgtcattacccaccccgacgttgccttctacgacttctacgaggcatggaacacgcccattgaggcggacaagaacagctacctgagcagccgcactggtatcctcgctcaggccgcgcccaacattggcccaatgatgtgggaggaaatcaagggtgccgacggtatcgtccgccagctgcaatggaccgcccgtgtcgagggtagctttgacacgcctaacgggcaggcgatgaccatctcgcagtacctcggccgcggcgcgacctcgcgcggccgtatgaccatcaccccttcgctgacgaccgtcgtctcggacgtgccgtacctcaaggacccgaacgataaggaggccgtcatccagggcatcgtcaacctgcagaacgccctcaaaaacgtcgccggcctgacctggacctaccccaactcgagcatcacaccgcgcgaatacgtcgataatatggtagtctcccctagcaaccggcgcgcaaaccactggatgggcacggccaaaatcggcaccgacgacggccgcctggccggcggctccgccgtcgtggacttgaacaccaaggtctacggcaccgacaacctctttgtcgtggacgcgtccatcttccccggcacgcccaccaccaatccctcggcgtacatcgtcacggctgcggagcatgcttcgcagaggatcttggggttggctgcgccgaagccggttgggaaatggggccagtgtggcgggcggcagtggacagggagcttccagtgcgtgagtgggacaaagtgtgaggtggtgaatgagtggtactcgcagtgcttgtag >C.thermophilum(SEQ ID NO: 8) atgaagcttctcagccgcgttggggccaccgccctagcggcgacgttgtccctgaaacaatgtgcagctcagatgaccgaagggacgtacacccatgaggctaccggtatcacgttcaagacatggactccttccgacggctcgactttcactttcggcttggccctccctggggacgcgctgacaaatgatgccaccgagtacattggtctcctgcgttgccaaatcaccgatccctcttcgcccggctactgtggcatctcccacggccagtccggccagatgacgcaggcgctgctgctggtcgcttgggccagcgaggatgtcgtctacacgtcgttccgctacgccaccggctacacactccccgagctctacacgggcgacgccaagctgacccagatcgcctcctcggtcagcggggacagcttcgaggtgctgttccgctgcgagaactgcttctcctgggaccagaacggcgccacgggcagtgtctcgaccagcaacggcgccctggttctcggctacgctgcctcgaagagtggtttgacgggcgccacgtgcccggacacggccgagtttggcttccacaacaatggtttcggacagtggggtgcagtgctcgagggtgcgacctcggactcgtatgaggagtgggctcagctggccactatcacgcccccgaccacctgcgatggcaacggccctggcgacaaggtgtgcgttccggctcccgaagacacgtatgattacatcgttgtcggcgccggcgccggcggcatcacggtcgccgacaagctcagcgaggccggccacaaggtcctccttatcgagaagggtcctccgtcgaccggcctgtggaacgggaccatgaagcccgagtggctcgagggtaccgacctcacccgcttcgacgtccccggtctgtgcaaccagatctgggtcgactctgccggcattgcctgcaccgataccgaccagatggcgggctgcgttctcggcggtggcaccgctgtcaatgctggtctgtggtggaagccccaccccgctgactgggacgacaacttccctcatggctggaagtcgagcgatctcgcggatgcgaccgagcgtgtcttcagccgcattcccggcacgtggcacccgtcgcaggatggcaaactgtaccgccaggagggcttcgaggtcatcagccagggcctggccaacgccggctggagggaagtcgacgccaaccaggagcccagcgagaagaaccgcacgtattcccacagtgtgttcatgttctcgggcggtgagcgcggcggccccctggcgacgtacctcgcctcggctgcccagcgcagcaacttcaacttgtgggtcaacacttcggtccggagggccatccgcaccggccccagggtcagtggcgtcgaactcgagtgccttgcggacggcggcttcaacggtactgtcaacctgaaggagggtggtggtgtcatcttttcggctggcgctttcggctcggccaagctgctccttcgcagcggcatcggtcctgaggaccagctcgagattgtggcgagctccaaggacggcgagaccttcatttccaagaatgattggatcaagctccccgtcggccataacctgatcgatcatctcaacaccgacctcattattactcacccggatgtcgttttctatgacttctacgcggcttgggacaatcccatcaccgaggacaaggaggcctacctgaactcgcggtccggcattctcgcccaagcggcgcccaacatcggccctctgatgtgggaggaagtcacgccatccgacggcatcacccgccagttccagtggacatgccgtgttgagggcgacagctccaagaccaactcgacccacgccatgaccctcagccagtatctcggccgtggcgtcgtctcgcgcggccggatgggcatcacttccgggctgaccacgacggtggccgagcacccgtacctgcacaacgacggcgacctggaggcggtgatccagggtatccagaacgtggtggacgcgctcagccaggtgcccgacctcgagtgggtgctcccgccgcccaacacgacggtggaggaatacgtcaacagcctgatcgtgtctccggctaaccgccgggccaaccactggatgggcacggccaagatgggcctcgatgacggccgctcgggcggctccgcggtcgtcgacctcaacacaaaggtgtatggcaccgacaacctgtttgtcgtcgacgcctccatcttccctggcatgtcgacgggcaacccgtcggctatgatcgtcatcgtggccgagcaggcggcccagcgcatcctgt ccctgcggtattag>S.bisbyi (SEQ ID NO: 10)atgctgttcaagctctcaaattggttgctagcgcttgcgctctttgttggcaatgtcgttgctcaactcgaggggcctaccccgtacacggatccagataccggcattgtctttcagtcctgggtcaatccagcagggaccctgaagtttggttacacttaccccgcaaatgctgctacggttgccgccacggaatttatcggtttcctggaatgccaaggggctggatggtgtagcgtctcactcggtggctccatgcttaacaagccgcttgttgttgcctaccctagtggcgatgaagtcctcgcttctttgaagtgggccacaggctacgcgaatccagagccttacggcggcaatcacaagctgtcccagatcagctcgtccgtcacctctgctggcttcagggtcgtctatcgatgtgagggatgtctcgcctggaactaccagggaattgagggagggagccccaccaatggtgcgtccatgcctatcggttgggcttacagcgcaagttctgtactcaacggggattgtgtggataacactgttctcattcaacatgacacctttggcaattatggcttcgtacctgatgaatcatctcttcgcacggagtacaatgactggacggagcttccgaccagggttgtcaggggagactgcggcggttccacaactacctcttcggtgccctcctcaacggcgcctcctcaaggtactggcataccggttcctactggcgcaagctatgactacatagttgttggctcgggtgctggaggtattcccattgcggataagcttaccgaggctggcaaaaaggttttgttgattgagaagggaccaccctcttctggtcgctacgatggaaagctaaagccgacgtggcttgagggaactaatctcacccgattcgatgtgcctggcctctgcaaccaaatatgggtcgactccgctggcattgcatgccgtgataccgatcagatggctggttgtgttcttggcggtggtactgctgtcaatgcaggtctatggtggaagcctaaccctattgattgggactataatttcccttcaggctggaagtcaagcgagatgataggcgcgacaaaccgtgtcttttcacgtattggtggtactactgttccttcgcaggacggaaagacctactatcagcaaggtttcaacgttctttccagcggtctcaaggctgcgggctggacatctgttagcctgaataacgcccctgcgcaaaggaaccgcacctatggtgctggccctttcatgttctctggtggagagcgaggtggacctttggccacctacctggccactgccaagaagagaggaaacttcgacctctggacgaatacccaagttaagcgtgtaattcgacagggaggtcatgttactggagtggaggtcgaaaactataacggtgatgggtacaagggcactgtcaaggtgactcctgtatctgggcgagttgtcctatctgctggtacctttggcagtgctaagcttttgctccgaagcggtatcggtcccaaggatcaactagctattgtcaagaactcgactgatggccctactatggcttccgagagggactggattaatcttcccgttggctacaacttggaggaccatactaacaccgacattgtcatctcccatccagatgtggtccattacgacttctatgaggcttggacagcgtcaatcgagtctgacaagactgcttatttgggcaagcgttctggcatcctcgctcaagccgcccccaacatcgggcctctcttctttgacgaagttcgcggtgctgacaacattgtccgctcaattcagtacactgctcgtgtggagggcaacagtgtggtccctaatggcaaggccatggtgatcagccagtaccttggtcgtggcgctgtttccaggggtcgaatgaccatctctcaaggtctcaatacgattgtttccaccgctccatacctctcaaacgtcaatgatctcgaggccgtcattaagagccttgagaacatagcgaacagcttgacgtcaaaggttaaaaacctcaagattgaatggcctgcctctggtacatccattcgcgatcacgtcacgaatatgcctctcgacccggccacccgccgagcgaatcattggattggcactaacaagatcggaaccaagaatggtcgactgacaggtggtgattccgtcgttgatttgaacactaaggtctatggtacagacaatctgtttgtggtcgatgcttctattttccctggcatggttacgaccaacccctcggcctacattgtaattgccgctgagcatgctgcatcgaagattctgagcctacctactgctaaggctgccgcgaagtacgaacaatgtggtggccttgaatataatggtaactttcagtgtgcgtctggattaacctgcacttggttaaacgactactactggcagtgtacttaa >N.crassa (SEQ ID NO: 12)atgaggaccacctcggcctttctcagcggcctggcggcggtggcttcattgctgtcgcccgccttcgcccaaaccgctcccaagaccttcactcatcctgataccggcattgtcttcaacacatggagtgcttccgattcccagaccaaaggtggcttcactgttggtatggctctgccgtcaaatgctcttactaccgacgcgactgaattcatcggttatctggaatgctcctccgccaagaatggtgccaatagcggttggtgcggtgtttctctcagaggcgccatgaccaacaatctactcattaccgcctggccttctgacggagaagtctacaccaatctcatgttcgccacgggttacgccatgcccaagaactacgctggtgacgccaagatcacccagatcgcgtccagcgtgaacgctacccacttcacccttgtctttaggtgccagaactgtttgtcatgggaccaagacggtgtcaccggcggcatttctaccagcaataagggggcccagctcggttgggtccaggcgttcccctctcccggcaacccgacttgccctacccagatcactctcagtcagcatgacaacggtatgggccagtggggagctgcctttgacagcaacattgccaatccctcttatactgcatgggctgccaaggccaccaagaccgttaccggtacttgcagtggtccagtcacgaccagtattgccgccactcctgttcccactggcgtttcttttgactacattgtcgttggtggtggtgccggtggtattcccgtcgctgacaagctcagcgagtccggtaagagcgtgctgctcatcgagaagggtttcgcttccactggtgagcatggtggtactctgaagcccgagtggctgaataatacatcccttactcgcttcgatgttcccggtctttgcaaccagatctggaaagactcggatggcattgcctgctccgataccgatcagatggccggctgcgtgctcggcggtggtaccgccatcaacgccggtctctggtacaagccctacaccaaggactgggactacctcttcccctctggctggaagggcagcgatatcgccggtgctaccagcagagccctctcccgcattccgggtaccaccactccttctcaggatggaaagcgctaccttcagcagggtttcgaggttcttgccaacggcctcaaggcgagcggctggaaggaggtcgattccctcaaggacagcgagcagaagaaccgcactttctcccacacctcatacatgtacatcaatggcgagcgtggcggtcctctagcgacttacctcgtcagcgccaagaagcgcagcaacttcaagctgtggctcaacaccgctgtcaagcgcgtcatccgtgagggcggccacattaccggtgtggaggttgaggccttccgcaacggcggctactccggaatcatccccgtcaccaacaccaccggccgcgtcgttctttccgccggcaccttcggcagcgccaagatccttctccgttccggcattggccccaaggaccagctcgaggtggtcaaggcctccgccgacggccctaccatggtcagcaactcgtcctggattgacctccccgtcggccacaacctggttgaccacaccaacaccgacaccgtcatccagcacaacaacgtgaccttctacgacttttacaaggcttgggacaaccccaacacgaccgacatgaacctgtacctcaatgggcgctccggcatcttcgcccaggccgcgcccaacattggccccttgttctgggaggagatcacgggcgccgacggcatcgtccgtcagctgcactggaccgcccgcgtcgagggcagcttcgagacccccgacggctacgccatgaccatgagccagtaccttggccgtggcgccacctcgcgcggccgcatgaccctcagccctaccctcaacaccgtcgtgtctgacctcccgtacctcaaggaccccaacgacaaggccgctgtcgttcagggtatcgtcaacctccagaaggctctcgccaacgtcaagggtctcacctgggcttaccctagcgccaaccagacggctgctgattttgttgacaagcaacccgtaacctaccaatcccgccgctccaaccactggatgggcaccaacaagatgggcaccgacgacggccgcagcggcggcaccgcagtcgtcgacaccaacacgcgcgtctatggcaccgacaacctgtacgtggtggacgcctcgattttccccggtgtgccgaccaccaaccctaccgcctacattgtcgtcgccgctgagcatgccgcggccaaaatcctggcgcaacccgccaacgaggccgttcccaagtggggctggtgcggcgggccgacgtatactggcagccagacgtgccaggcgccatataagtgcgagaagcagaatgattggtattggcagtgtgtgtag

1.-26. (canceled)
 27. A cellobiose dehydrogenase (CDH) further definedas a modified CDH of Myriococcum thermophilum or a CDH isolated fromChaetomium atrobrunneum, Corynascus thermophilus, Hypoxylonhaematostroma, Neurospora crassa or Stachybotris bisbyi and havingglucose oxidation activity at a pH of 7.4 or above.
 28. The cellobiosedehydrogenase of claim 27, further defined as a CDH of Chaetomiumatrobrunneum, Hypoxylon haematostroma, or Stachybotris bisbyi or amodified CDH of Myriococcum thermophilum.
 29. The cellobiosedehydrogenase of claim 27, further defined as comprising an amino acidsequence that is at least 50% identical to SEQ ID NO: 3, SEQ ID NO: 5,SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO:
 11. 30. The cellobiosedehydrogenase of claim 29, further defined as comprising an amino acidsequence that is at least 75% identical to SEQ ID NO: 3, SEQ ID NO: 5,SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO:
 11. 31. The cellobiosedehydrogenase of claim 30, further defined as comprising an amino acidsequence that is at least 90% identical to SEQ ID NO: 3, SEQ ID NO: 5,SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO:
 11. 32. The cellobiosedehydrogenase of claim 31, further defined as comprising an amino acidsequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 orSEQ ID NO:
 11. 33. The cellobiose dehydrogenase of claim 27, furtherdefined as a modified CDH of Myriococcum thermophilum, comprising aflavin and a haem domain, wherein electron transfer from the flavin tothe haem domain is increased by a modification as compared to wild typeCDH of Myriococcum thermophilum.
 34. The cellobiose dehydrogenase ofclaim 33, further defined as comprising an amino acid sequence that isat least 50% identical to amino acids 22 to 828 of SEQ ID NO: 1 and atleast one amino acid substitution, deletion, or insertion to thesequence of SEQ ID NO: 1 that increases electron transfer from theflavin to the haem domain as compared to wild-type CDH of M.thermophilum of SEQ ID NO:
 1. 35. The cellobiose dehydrogenase of claim33, further defined as comprising an amino acid sequence that is atleast 75% identical to amino acids 22 to 828 of SEQ ID NO: 1 and the atleast one amino acid substitution, deletion, or insertion to thesequence of SEQ ID NO:
 1. 36. The cellobiose dehydrogenase of claim 33,further defined as comprising an amino acid sequence that is at least90% identical to amino acids 22 to 828 of SEQ ID NO: 1 and the at leastone amino acid substitution, deletion, or insertion to the sequence ofSEQ ID NO:
 1. 37. The cellobiose dehydrogenase of claim 33, furtherdefined as comprising an amino acid sequence of amino acids 22 to 828 ofSEQ ID NO: 1 and the at least one amino acid substitution, deletion, orinsertion to the sequence of SEQ ID NO:
 1. 38. The cellobiosedehydrogenase of claim 33, wherein the electron transfer is increased byincreasing electrostatic interactions between the flavin and the haemdomain.
 39. The cellobiose dehydrogenase of claim 33, wherein themodification is a modification of the haem domain of any one of aminoacids 90-100, 115-124, and/or 172-203 and/or of the flavin domain of anyone of amino acids 311-333, 565-577, 623-625, 653-664, and/or 696-723 ofSEQ ID NO: 1, or any combination thereof.
 40. The cellobiosedehydrogenase of claim 39, wherein the modification is a modification ofthe haem domain of any one of amino acids 176, 179-182, 195, 196, 198,and/or 201 and/or of the flavin domain of any one of amino acids 318,325, 326, 328, 568, 571, 574, 575, 624, 654, 663, 702, 709, 712, and/or717 of SEQ ID NO: 1, or any combination thereof.
 41. The cellobiosedehydrogenase of claim 33, wherein the modification is an increase ofpositive charge in amino acids 172-203 and/or a decrease of a negativecharge of amino acids 565-577 of SEQ ID NO: 1, or any combinationthereof.
 42. The cellobiose dehydrogenase of claim 41, wherein themodification is an increase of positive charge in amino acid 181 and/ora decrease of a negative charge of amino acids 568 and/or 571 and/or 574of SEQ ID NO: 1, or any combination thereof.
 43. The cellobiosedehydrogenase of claim 42, wherein the modification is a D181K or D181Rmutation and/or a D568S and/or E571S and/or D574S mutation.
 44. Thecellobiose dehydrogenase of claim 27, wherein the glucose oxidationactivity is a glucose dehydrogenase activity.
 45. The cellobiosedehydrogenase of claim 27, wherein the activity is an electrocatalyticoxidation of glucose.
 46. A nucleic acid molecule encoding a cellobiosedehydrogenase of claim 27 comprising: a nucleotide sequence of SEQ IDNOs: 4, 6, 8, 10 or 12; or the open reading frame of SEQ ID NOs: 4, 6,8, 10 or 12; or a nucleotide sequence with at least 50% identity to SEQID NOs: 2, 4, 6, 8, 10 or 12 or the open reading frame of SEQ ID NOs: 2,4, 6, 8, 10 or 12, further comprising a nucleotide mutation,substitution, deletion or insertion; or a nucleotide sequence thathybridizes with any one of SEQ ID NOs: 2, 4, 6, 8, 10 or 12 understringent conditions.
 47. The nucleic acid of claim 46, further definedas comprising a nucleotide sequence with at least 50% identity to SEQ IDNOs: 2, 4, 6, 8, 10 or 12 or the open reading frame of SEQ ID NOs: 2, 4,6, 8, 10 or
 12. 48. The nucleic acid of claim 46, further defined ascomprising a nucleotide sequence with at least 65% identity to SEQ IDNOs: 2, 4, 6, 8, 10 or 12 or the open reading frame of SEQ ID NOs: 2, 4,6, 8, 10 or
 12. 49. The nucleic acid of claim 46, further defined ascomprising a nucleotide sequence with at least 80% identity to SEQ IDNOs: 2, 4, 6, 8, 10 or 12 or the open reading frame of SEQ ID NOs: 2, 4,6, 8, 10 or
 12. 50. The nucleic acid of claim 46, further defined ascomprising a nucleotide sequence with at least 95% identity to SEQ IDNOs: 2, 4, 6, 8, 10 or 12 or the open reading frame of SEQ ID NOs: 2, 4,6, 8, 10 or
 12. 51. The nucleic acid of claim 46, further defined ascomprising a nucleotide sequence with at least 99% identity to SEQ IDNOs: 2, 4, 6, 8, 10 or 12 or the open reading frame of SEQ ID NOs: 2, 4,6, 8, 10 or
 12. 52. The nucleic acid of claim 46, further defined ascomprising a nucleotide sequence of SEQ ID NOs: 2, 4, 6, 8, 10 or 12 orthe open reading frame of SEQ ID NOs: 2, 4, 6, 8, 10 or
 12. 53. Anelectrode comprising an immobilized cellobiose dehydrogenase of claim 27and having at least 10% glucose, lactose or cellobiose oxidizingactivity at a pH of 7.4 as compared to maximal activity at a lower pH asdetermined by a cyt c assay.
 54. The electrode of claim 53, furtherdefined as comprising an immobilized cellobiose dehydrogenase and havingat least 14% glucose, lactose or cellobiose oxidizing activity at a pHof 7.4 as compared to maximal activity at a lower pH as determined bythe cyt c assay.
 55. The electrode of claim 53, further defined ascomprising an immobilized cellobiose dehydrogenase and having at least20% glucose, lactose or cellobiose oxidizing activity at a pH of 7.4 ascompared to maximal activity at a lower pH as determined by the cyt cassay.
 56. The electrode of claim 53, further defined as comprising animmobilized cellobiose dehydrogenase and having at least 30% glucose,lactose or cellobiose oxidizing activity at a pH of 7.4 as compared tomaximal activity at a lower pH as determined by the cyt c assay.
 57. Theelectrode of claim 53, wherein the glucose oxidation activity at pH 7.4is at least 0.5 U/mg cellobiose dehydrogenase.
 58. The electrode ofclaim 53, wherein the cellobiose dehydrogenase has a Km value for aglucose oxidation reaction at pH 7.4 of below 1M.
 59. The electrode ofclaim 53, wherein the cellobiose dehydrogenase is immobilized byadsorption or complex formation and/or wherein the immobilizedcellobiose dehydrogenase is cross-linked to increase stability oractivity.
 60. The electrode of claim 59, wherein the cellobiosedehydrogenase is immobilized by complex formation via an additionalcomplexing linker, covalent or ionic linkage and/or wherein theimmobilized cellobiose dehydrogenase is cross-linked by at least onebifunctional agent.
 61. An electrochemical cell comprising an electrodeof claim 53 as an anodic element and a cathodic element.
 62. Theelectrochemical cell of claim 61, comprising a glucose containingsolution as anodic fluid.
 63. The electrochemical cell of claim 61,further defined as comprising a solution of at least pH 6.0 as anodicfluid.
 64. The electrochemical cell of claim 63, further defined ascomprising a solution of at least pH 6.5 as anodic fluid.
 65. Theelectrochemical cell of claim 64, further defined as comprising asolution of at least pH 7.2 as anodic fluid.
 66. A method of detectingand/or quantifying glucose in a sample comprising: providing acellobiose dehydrogenase of claim 27; contacting a fluid sample having apH of at least 6.5 with the cellobiose dehydrogenase; and detecting anoxidation, if any, of glucose in the sample by the cellobiosedehydrogenase.
 67. The method of claim 66, wherein the oxidation isdetected electrochemically.
 68. The method of claim 67, wherein theoxidation is detected with an immobilized cellobiose dehydrogenase on anelectrode.
 69. The method of claim 66, wherein the cellobiosedehydrogenase has at least 10% glucose, lactose or cellobiose oxidizingactivity at a pH of 7.4 as compared to maximal activity at a lower pH asdetermined by a cyt c assay.
 70. The method of claim 69, wherein thecellobiose dehydrogenase has at least 20% glucose, lactose or cellobioseoxidizing activity at a pH of 7.4 as compared to maximal activity at alower pH as determined by a cyt c assay.
 71. The method of claim 70,wherein the cellobiose dehydrogenase has at least 30% glucose, lactoseor cellobiose oxidizing activity at a pH of 7.4 as compared to maximalactivity at a lower pH as determined by a cyt c assay.